Compositions for improving the health and appearance of skin

ABSTRACT

Provided herein are microalgal skin care compositions and methods of improving the health and appearance of skin. Also provided are methods of using polysaccharides for applications such as topical personal care products, cosmetics, and wrinkle reduction compositions. The invention also provides novel decolorized microalgal compositions useful for improving the health and appearance of skin. The invention also includes insoluble polysaccharide particles for application to human skin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application No. 60/961,173, filed Jul. 18, 2007,which is incorporated herein by reference in its entirety for allpurposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a sequence listing as shown in pages 1-70 ofthe Sequence Listing attached hereto.

FIELD OF THE INVENTION

The invention resides in the fields of health and beauty, aquaculture,and genetic engineering.

BACKGROUND OF THE INVENTION

Carbohydrates have the general molecular formula CH₂O, and thus wereonce thought to represent “hydrated carbon”. However, the arrangement ofatoms in carbohydrates has little to do with water molecules. Starch andcellulose are two common carbohydrates. Both are macromolecules withmolecular weights in the hundreds of thousands. Both are polymers; thatis, each is built from repeating units, monomers, much as a chain isbuilt from its links.

Three common sugars share the same molecular formula: C₆H₁₂O₆. Becauseof their six carbon atoms, each is a hexose. Glucose is the immediatesource of energy for cellular respiration. Galactose is a sugar in milk.Fructose is a sugar found in honey. Although all three share the samemolecular formula (C₆H₁₂O₆), the arrangement of atoms differs in eachcase. Substances such as these three, which have identical molecularformulas but different structural formulas, are known as structuralisomers. Glucose, galactose, and fructose are “single” sugars ormonosaccharides.

Two monosaccharides can be linked together to form a “double” sugar ordisaccharide. Three common disaccharides are sucrose, common table sugar(glucose+fructose); lactose, the major sugar in milk(glucose+galactose); and maltose, the product of starch digestion(glucose+glucose). Although the process of linking the two monomers iscomplex, the end result in each case is the loss of a hydrogen atom (H)from one of the monosaccharides and a hydroxyl group (OH) from theother. The resulting linkage between the sugars is called a glycosidicbond. The molecular formula of each of these disaccharides isC₁₂H₂₂O₁₁═2 C₆H₁₂O₆—H₂O. All sugars are very soluble in water because oftheir many hydroxyl groups. Although not as concentrated a fuel as fats,sugars are the most important source of energy for many cells.

SUMMARY OF THE INVENTION

The present invention relates to polysaccharides and biomass producedfrom microalgae or other microorganisms. Representative polysaccharidesinclude those present in the cell wall of microalgae as well as secretedpolysaccharides, or exopolysaccharides. In addition to thepolysaccharides themselves, such as in an isolated, purified, orsemi-purified form, the invention includes a variety of compositionscontaining one or more microalgal polysaccharides as disclosed herein.The compositions include nutraceutical, cosmeceutical, industrial andpharmaceutical compositions which may be used for a variety ofindications and uses as described herein. Other compositions includethose containing one or more microalgal polysaccharides and a suitablecarrier or excipient for topical or oral administration.

The present invention also relates to decolorized microalgae forformulation in skin care products as a composition of the disclosedinvention. The invention thus provides highly desirable compositions ofmicroalgal cells that do not stain human skin with red or green pigmentsbut still provide delivery or high value cosmeceutical ingredients suchas carotenoids, polyunsaturated fatty acids, moisturizingpolysaccharides, superoxide dismutase, and other components.

The invention provides the insight that combinations of high lightirradiance and limiting levels of nitrogen-containing compounds in theculture media allow production of biomass high incosmeceutical/nutraceutical value but do not contain substantial amountsof pigments that stain human skin when applied as part of a skin careformulation. In addition, antioxidant, moisturizing polysaccharides areproduced at higher levels in microalgae cells such as those of the genusPorphyridium under high light/low nitrogen conditions. The inventionprovides compositions of Porphyridium biomass that are substantiallyfree of red coloration and contain higher amounts of exopolysaccharidethan cells containing significant amounts of red coloration that aregrown under nitrogen-replete conditions.

In one aspect, the disclosed invention includes a composition comprisingcells of the genus Porphyridium, wherein an aqueous extract of thecomposition contains a reduced level of red pigmentation, or a reducedabsorbance at 545 nm, relative to the same cells grown under differentconditions. In some embodiments, the extract contains no more than about75% to no more than about 5% of the absorbance per gram at 545 nmcompared to an extract of cells of the same species grown in aphotobioreactor in ATCC 1495 artificial seawater (ASW) media in thepresence of 50 microeinsteins of light per second per square meter. Inother embodiments, the composition comprises a carrier and/or apreservative suitable for topical administration. In additionalembodiments, the carrier is suitable for human topical administration.

The invention further relates to methods of producing or preparingmicroalgal polysaccharides. In some disclosed methods, exogenous sugarsare incorporated into the polysaccharides to produce polysaccharidesdistinct from those present in microalgae that do not incorporateexogenous sugars. The invention also includes methods of trophicconversion and recombinant gene expression in microalgae. In somemethods, recombinant microalgae are prepared to express heterologousgene products, such as mammalian proteins as a non-limiting example,while in other embodiments, the microalgae are modified to produce moreof a small molecule already made by microalgae in the absence of geneticmodification.

The invention further relates to methods of growing, producing andpreparing microalgal biomass. In some disclosed methods, excess light isprovided as one method of removing pigmentation. In other methods,reducing the amount of nitrogen provided to microalgae cells in cultureis provided as one method of removing pigmentation. In other methods,increased light irradiance combined with culture media containinglimiting amounts of nitrogen are used to reduce and/or eliminate red orgreen pigmentation. Additionally, the invention provides decolorizedstrains produced through chemical mutagenesis or gene insertion/deletionmethods that are used to generate biomass for skin care products.

In another aspect, the invention relates to compositions for topicalapplication, such as a composition for application to human skincomprising a polysaccharide isolated from cells of the genusPorphyridium. In some embodiments, the composition comprises apolysaccharide that is part of a microalgal cell, or a homogenatethereof. In other embodiments, the polysaccharide is contained withinmicroalgal cells, or a homogenate thereof, which is essentially free, orcompletely free, of red coloration. Thus, a composition of the disclosedinvention may also be essentially free, or completely free, of redcoloration. Non-limiting examples include compositions comprising lessthan about 15 milligrams, less than about 1 milligram, or less thanabout 0.1 milligrams of phycoerythrin per dry gram of cells in thecomposition.

In additional embodiments, the composition is that of a cosmetic orother skin care product. Such products may contain one or moremicroalgal polysaccharides, or a microalgal cell homogenate, a topicalcarrier, and/or a preservative. In some embodiments, the carrier may beany carrier suitable for topical application, such as, but not limitedto, use on human skin or human mucosal tissue. In other embodiments, thecomposition may contain a purified microalgal polysaccharide, such as anexopolysaccharide, and a topical carrier. Exemplary carriers includeliposome formulation, biodegradable microcapsule, lotion, spray,aerosol, dusting powder, biodegradable polymer, mineral oil, liquidpetroleum, white petroleum, propylene glycol, polyoxyethylenepolyoxypropylene compound, emulsifying wax, sorbitan monostearate,polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water. Exemplary preservatives includediiodomethyl-p-tolylsulfone, 2-Bromo-2-nitropropane-1,3-diol, cis isomer1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride,glutaraldehyde, 4,4-dimethyl oxazolidine, 7-Ethylbicyclooxazolidine,methyl paraben, sorbic acid, Germaben II, and disodium EDTA.

As a cosmeceutical, the composition may contain a microalgalpolysaccharide or homogenate and other component material found incosmetics. In some embodiments, the component material may be that of afragrance, a colorant (e.g. black or red iron oxide, titanium dioxideand/or zinc oxide, etc.), a sunblock (e.g. titanium, zinc, etc.), and amineral or metallic additive.

In other aspects, the invention includes methods of preparing orproducing a microalgal polysaccharide. In some aspects relating to anexopolysaccharide, the invention includes methods that separate theexopolysaccharide from other molecules present in the medium used toculture exopolysaccharide producing microalgae. In some embodiments,separation includes removal of the microalgae from the culture mediumcontaining the exopolysaccharide, after the microalgae has been culturedfor a period of time. Of course the methods may be practiced withmicroalgal polysaccharides other than exopolysaccharides. In otherembodiments, the methods include those where the microalgae was culturedin a bioreactor, optionally where a gas is infused into the bioreactor.

In one embodiment, the invention includes a method of producing anexopolysaccharide, wherein the method comprises culturing microalgae ina bioreactor, wherein gas is infused into the bioreactor; separating themicroalgae from culture media, wherein the culture media contains theexopolysaccharide; and separating the exopolysaccharide from othermolecules present in the culture media.

The microalgae of the invention may be that of any species, includingthose listed in Tables 1A and 1B herein. In some embodiments, themicroalgae is a red algae, such as the red algae Porphyridium, which hastwo known species (Porphyridium sp. and Porphyridium cruentum) that havebeen observed to secrete large amounts of polysaccharide into theirsurrounding growth media. In other embodiments, the microalgae is of agenus selected from Rhodella, Chlorella, and Achnanthes. Non-limitingexamples of species within a microalgal genus of the invention includePorphyridium sp., Porphyridium cruentum, Porphyridium purpureum,Porphyridium aerugineum, Rhodella maculata, Rhodella reticulata,Chlorella autotrophica, Chlorella stigmatophora, Chlorella capsulata,Achnanthes brevipes and Achnanthes longipes.

In some embodiments, a polysaccharide preparation method is practicedwith culture media containing over 26.7, or over 27, mM sulfate (ortotal SO₄ ²⁻). Non-limiting examples include media with more than about28, more than about 30, more than about 35, more than about 40, morethan about 45, more than about 50, more than about 55, more than about60, more than about 65, more than about 70, more than about 75, morethan about 80, more than about 85, more than about 90, more than about95, more than about 100 mM sulfate, and in some instances more than 250mM, more than 400 mM, more than 550 mM, and more than 750 mM, more than900 mM, more than 1M, and more than 2 mM sulfate. Sulfate in the mediamay be provided in one or more of the following forms: Na₂SO₄.10H₂O,MgSO₄.7H₂0, MnSO₄, and CuSO₄.

Other embodiments of the method include the separation of anexopolysaccharide from other molecules present in the culture media bytangential flow filtration. Alternatively, the methods may be practicedby separating an exopolysaccharide from other molecules present in theculture media by alcohol precipitation. Non-limiting examples ofalcohols to use include ethanol, isopropanol, and methanol.

In other embodiments, a method may further comprise treating apolysaccharide or exopolysaccharide with a protease to degradepolypeptide (or proteinaceous) material attached to, or found with, thepolysaccharide or exopolysaccharide. The methods may optionally compriseseparating the polysaccharide or exopolysaccharide from proteins,peptides, and amino acids after protease treatment.

In other embodiments, a method of formulating a cosmeceuticalcomposition is disclosed. As one non-limiting example, the compositionmay be prepared by adding separated polysaccharides, orexopolysaccharides, to homogenized microalgal cells before, during, orafter homogenization. Both the polysaccharides and the microalgal cellsmay be from a culture of microalgae cells in suspension and underconditions allowing or permitting cell division. The culture mediumcontaining the polysaccharides is then separated from the microalgalcells followed by 1) separation of the polysaccharides from othermolecules in the medium and 2) homogenization of the cells.

Other compositions of the invention may be formulated by subjecting aculture of microalgal cells and soluble exopolysaccharide to tangentialflow filtration until the composition is substantially free of salts.Alternatively, a polysaccharide is prepared after proteolysis ofpolypeptides present with the polysaccharide. The polysaccharide and anycontaminating polypeptides may be that of a culture medium separatedfrom microalgal cells in a culture thereof. In some embodiments, thecells are of the genus Porphyridium.

In a further aspect, the disclosed invention includes a compositioncomprising particulate polysaccharides. The polysaccharides may be fromany microalgal source, and with any level of sulfation, as describedherein. The composition may be sterile or substantially free ofendotoxins and/or proteins in some embodiments. In other embodiments,the composition further comprises hyaluronic acid or another agentsuitable or desirable for treatment of skin. The particles in someembodiments are generated by first purifying the polysaccharide awayfrom biomass, then drying the purified polysaccharide into a film, andthen homogenizing and/or grinding the film into smaller particles.

In some embodiments, the polysaccharides are in the form of a purifiedmaterial that was dried to be completely or partially insoluble inwater. Preferably the purified material has been separated from cellbiomass, for example as described in Example 2. In such purified formthe polysaccharide is at least 50% polysaccharide by weight, and morepreferably above 75% polysaccharide by weight. In some embodiments thepolysaccharide is associated with one or more species of protein thatcan be endogenous to the microalgae source, and alternatively can be afusion protein that is partially endogenous to the microalgae source asdescribed herein. In some embodiments, the dried polysaccharideparticles are in mixture with a non-aqueous solvent or material. Inother embodiments, the dried polysaccharide particles are partiallysoluble such that they are from less than about 70% to less than about1% soluble in water.

In additional embodiments, the polysaccharide particles increase involume, or swell, on contact with water or water vapor. Thus the volumeof the polysaccharide particles increases compared to its anhydrous orpartially hydrated volume before exposure to the water or water vapor.In some embodiments, the particles increase in volume by an amountselected from at least about 5% to at least about 5000%.

In some embodiments, the polysaccharide compositions described hereinfurther comprise at least one ingredient selected from the groupconsisting of beta carotene, lutein, astaxanthin, vitamin C, vitamin E,vitamin A, coenzyme Q10, a peptide, an aceylated peptide, oil solubleα-hydroxy acid, an alkyl lactate, and salicylic acid. In some cases, thecompositions comprise micronized particles containing the polysaccharideand the at least one other ingredient. In some cases, the particles areof a substantially uniform size. In some embodiments, the algalpolysaccharide and the at least one ingredient have been subjected toheating, drying and homogenization to form particles comprising bothalgal polysaccharides and the at least one ingredient. In some cases,the particles comprising both algal polysaccharides and the at least oneingredient have been processed to a substantially uniform size.

The disclosed invention further includes methods for the preparation ormanufacture of the dried polysaccharide particles. In some embodiments,the method comprises formulating particles of polysaccharide materialinto a non-aqueous material. The particles may be formed from a film ofdried polysaccharide material, wherein at least a portion (or someproportion) of the film has been made completely or partially insolublein water. Optionally, the particles are formed by homogenization of thefilm into particulate form.

In some cases, the film is formed by heating a suspension ofpolysaccharide material until all or part of the film is insoluble. Theheating may be of an aqueous suspension of the material to remove waterfrom the suspension. Of course the polysaccharide in the suspension maybe from any microalgal source as described herein. Optionally, thepolysaccharide in the suspension has been isolated from microalgalbiomass. Optionally, the polysaccharide in the suspension has beenisolated from supernatant of a culture of microalgae.

The disclosed invention thus includes a method of preparing ormanufacturing a composition for topical application, such as forimproving the appearance of skin. The method may comprise 1) drying anaqueous suspension of a polysaccharide isolated from microalgae to asolid film, wherein at least some proportion of the film has been madecompletely or partially insoluble in water; 2) homogenizing the filminto particles; and optionally 3) formulating the particles into anon-aqueous material. In some embodiments, the homogenizing is via amethod selected from jet milling, ball milling, Retsch® milling, pinmilling and milling in a Quadro® device. Optionally, the formulating ofthe particles is into the non-aqueous phase of an oil-in-water emulsion,such as an emulsion suitable for topical application. The non-aqueousphase may comprise an oil suitable for topical application, such ashexadecanoic acid as a non-limiting example. In other cases, theformulating of the particles is into a carrier suitable for topicaladministration as described herein. In some embodiments, the particlesmay be relatively uniform in size or may range in size, but in manyembodiments, the particles have an average size between about 400 and0.1 microns.

The formation of a solid film may be by heating performed between about40 and about 180 degrees Celsius. In other embodiments, the heating isperformed in two parts. The first part may comprise heating asuspension, optionally aqueous, of polysaccharide material to no morethan about 60 to about 100° C. for a time period sufficient to form orproduce a solid film. This may be followed by a second heating of thesolid film for a (second) time period sufficient to reach no more thanabout 148 to about 160° C. In one embodiment the first heating is in thepresence of air, which may be optionally combined with the secondheating (of the solid film) being in at least a partial vacuum or in ahigh vacuum. Of course the second heating under reduced pressure may beused independent of the first heating in the presence of air. In otherembodiments the heating is done in a single step, either in the presenceof air or in the presence of a partial or full vacuum.

In some alternative embodiments, a method to render the polysaccharidematerial insoluble is selected from chemical cross-linking, chemicaldehydration through displacement of bound water by an alcohol,precipitation from solution using an alcohol or a ketone or pH, andcoating of particles by microencapsulation.

In an additional aspect, the disclosed invention includes a method oftopically applying a composition comprising polysaccharides inparticulate form. In some embodiments, the application is to skin, suchas to mammalian or human skin. Alternatively, the application is to lipsor wrinkles on human skin, or by injection into skin or a skin tissue.In many embodiments, the application is to improve the appearance ofskin.

In additional embodiments, a polysaccharide containing composition(optionally with polysaccharides in particulate form) may be used in amethod of cosmetic enhancement. In one embodiment, a method may includeinjecting a polysaccharide produced by microalgae into mammalian skin.Preferably the polysaccharide is sterile and free of protein.

In further embodiments, a method to treat skin, such as mammalian orhuman skin, is disclosed. In some embodiments, the method is for thetreatment of human facial skin or a tissue thereof. Such methods includea method to stimulating collagen synthesis, stimulating elastinsynthesis, or inhibiting collagenase activity in such skin by applying adisclosed composition of the invention to the skin. Additional methodsinclude a method to reduce the signs of aging or reduce the appearanceof aging in human skin by applying a composition of the disclosedinvention to the skin. Non-limiting examples of a sign of aging or anappearance of aging include wrinkles, such as those on the forehead oraround the eyes and/or lips, and liver spots (yellowish-brown flat spotsthat appear as large freckles). In some embodiments, a sign orappearance of aging is associated with reactive oxygen species (ROS)formation and/or activity in the skin. The use of a composition may thusbe based in part on the insight that the disclosed polysaccharidespossess anti-oxidant activity, and that further the high sulfatedpolysaccharides wherein the percent of sulfur by weight is above 4.75%.

Additional embodiments include the use of a polysaccharide containingcomposition in a method of reducing the effects of ultraviolet (UV)light or radiation, such as that present in sunlight, on skin or a skintissue. One non-limiting example is a method of shielding mammalian skinfrom UV light. The method may comprise applying a composition of thedisclosed invention to skin or a skin tissue in an effective orsufficient amount to shield, at least in part, the skin from UVradiation. In an alternative embodiment, a composition of the inventionmay be applied in an effective or sufficient amount to treat skin thathas been damaged by UV radiation. An additional non-limiting example isa method of for treating skin to reduce the risk of skin cancer inducedby sunlight or UV radiation. The method may comprise applying acomposition of the invention in an effective or sufficient amount toreduce the risk of UV or sunlight induced skin cancer.

An additional non-limiting example is a method of for treating skin toreduce the risk of skin cancer induced by sunlight or UV radiation thatcauses erythema. Erythema is redness of the skin caused by increasedblood flow to the capillaries. A subject can assess the effective amountof microalgal materials sufficient to treat erythema using methods knownin the art. See for example J. Invest. Dermatol., 117 (5); 1318-1321(2001).

In addition to the above, application of a composition of the inventionto human skin may be used in a method of reducing reactive oxygenspecies (ROS) in the skin or a skin tissue. This is based in part on theinsight that the disclosed polysaccharides possess anti-oxidantactivity. In some embodiments, the method is used to prevent or treat adisease or unwanted condition associated with ROS or oxidative stress.Non-limiting examples of such a disease or unwanted condition includereducing inflammation or irritation of the skin. In some embodiments,the polysaccharide composition comprises one or more other agents orcompounds with anti-oxidant activity. In further embodiments, the methodmay be used to lower the level of ROS, or reduce or decrease the amountof damage caused by ROS in skin or a skin tissue. The amount of thecomposition may be any that is effective or sufficient to produce adesired improvement or therapeutic benefit.

In other aspects, the present invention is directed to a method ofreducing fine lines and/or wrinkles on human skin, a method of inducinga feel of tightening human skin, a method of reducing transepidermalwater loss in human skin, a method of moisturizing human skin, and/or amethod of increasing elasticity of human skin. Each method comprisesadministration of a composition, as disclosed herein, to human skin inan amount and at a frequency sufficient to impart the desiredcharacteristics. The various methods and/or compositions can be combinedto provide methods or compositions suitable for imparting multiplecharacteristics simultaneously.

The details of additional embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features andadvantages of the invention will be apparent from the drawings anddetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows precipitation of 4 liters of Porphyridium cruentumexopolysaccharide using 38.5% isopropanol. (a) supernatant; (b) additionof 38.5% isopropanol; (c) precipitated polysaccharide; (d) separatingstep.

FIG. 2 shows growth of Porphyridium sp, and Porphyridium cruentum cellsgrown in light in the presence of various concentrations of glycerol.Glycerol concentrations in the table indicate ATCC 1495 mediasupplemented with the given percentage of glycerol. The cell counts inthe table are X 106 cells/mL. The percent change was calculated as thedifference in the number of cells/mL at a given glycerol concentrationcompared to the number of cells/mL at a glycerol concentration of zero.

FIG. 3 shows Porphyridium sp. cells grown in the dark in the presence ofvarious concentrations of glycerol.

FIG. 4 shows levels of solvent-accessible polysaccharide in Porphyridiumsp. homogenates subjected to various amounts of physical disruption fromSonication Experiment 1.

FIG. 5 shows levels of solvent-accessible polysaccharide in Porphyridiumsp. homogenates subjected to various amounts of physical disruption fromSonication Experiment 2.

FIG. 6 shows protein concentration measurements of autoclaved,protease-treated, and diafiltered exopolysaccharide.

FIG. 7 shows various amounts and ranges of amounts of compounds foundper gram of cells in cells of the genus Porphyridium.

FIG. 8 shows Porphyridium sp. cultured on agar plates containing variousconcentrations of zeocin.

FIG. 9 shows cultures of nitrogen-replete (flask 1) and nitrogen-starved(flask 2) Porphyridium cruentum. The culture media in flask 1 is asdescribed in Example 1. The culture media in flask 2 is as described inExample 1 except that the culture media contained no Tris and 0.125 g/Lpotassium nitrate, pH 7.6. Flasks 1 and 2 were inoculated with identicalamounts of deep red colored cells taken from a culture grown in ATCC1495ASW media. The cells in flask 2 are substantially free of redcoloration.

FIG. 10 shows lyophilized biomass from nitrogen-replete (panel (a)) andnitrogen-starved (panel (b)) Porphyridium cruentum cells. The culturemedia used to grow the cells shown in panel 1 was as described inExample 1. The culture media used to grow the cells shown in panel 2 wasas described in Example 1 except that the culture media contained noTris and 0.125 g/L potassium nitrate, pH 7.6. The lyophilized biomass inpanel 2 is substantially free of red coloration. Panel (c) shows threecolonies of wild-type Porphyridium sp. (top of plate) that exhibit fullred pigmentation, as well as three mutagenized colonies of Porphyridiumsp. that are substantially free of red coloration of plate).

FIG. 11( a) shows UVB-induced TT dimer formation in the presence andabsence of microalgae-derived materials. FIG. 11( b) shows secretion ofprocollagen by human fibroblasts in the presence and absence ofmicroalgae-derived materials.

FIG. 12( a) shows secretion of elastin by human fibroblasts in thepresence and absence of microalgae-derived materials. FIG. 12( b) showsinhibition of PMN migration in the presence and absence ofmicroalgae-derived materials.

FIG. 13( a) shows secretion of IL1-α in the presence and absence ofmicroalgae-derived materials. FIG. 13( b) shows secretion of gammainterferon in the presence and absence of microalgae-derived materials.

FIG. 14( a) shows PCR genotyping of two Porphyridium transformants forthe ble antibiotic resistance transgene. FIG. 14( b) shows PCRgenotyping of two Porphyridium transformants for the endogenousglycoprotein gene promoter. FIG. 14( c) shows PCR genotyping of onePorphyridium transformant for an exogenous gene encoding a recoded humanGLUT1 transporter.

FIG. 15 shows a Southern blot indicating chromosomal integration of anexogenous gene encoding a recoded human GLUT1 transporter.

FIG. 16 shows insoluble and soluble polysaccharide bead preparations.

FIG. 17( a) shows swelling of polysaccharide beads over time. FIG. 17(b) shows percentages of polysaccharide in the insoluble gel phase overtime.

FIG. 18 shows PBMC proliferation in the presence and absence ofmicroalgae-derived materials.

FIG. 19 shows polysaccharide protection from UV damage to human skinmodels.

FIG. 20 shows transformation of exogenous genes into Porphyridium sp.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

“Active in microalgae” means a nucleic acid that is functional inmicroalgae. For example, a promoter that has been used to drive anantibiotic resistance gene to impart antibiotic resistance to atransgenic microalgae is active in microalgae. Nonlimiting examples ofpromoters active in microalgae are promoters endogenous to certain algaespecies and promoters found in plant viruses.

“ARA” means Arachidonic acid.

“Associates with” means, within the context of a polysaccharide bindingfusion protein, one molecule binding to another molecule. Affinity andselectivity of binding can vary when a polysaccharide and apolysaccharide binding protein are in association with each other.

“Axenic” means a culture of an organism that is free from contaminationby other living organisms.

The term “biomass” refers to material produced by growth and/orpropagation of cells. Biomass may contain cells and/or intracellularcontents as well as extracellular material. Extracellular materialincludes, but is not limited to, compounds secreted by a cell.

“Bioreactor” means an enclosure or partial enclosure in which cells arecultured in suspension.

“Carrier suitable for topical administration” means a compound that maybe administered, together with one or more compounds of the presentinvention, and which does not destroy the activity thereof and isnontoxic when administered in concentrations and amounts sufficient todeliver the compound to the skin or a mucosal tissue.

“Cellulosic material” means the products of digestion of cellulose,including glucose and xylose, and optionally additional compounds suchas disaccharides, oligosaccharides, lignin, furfurals and othercompounds. Nonlimiting examples of sources of cellulosic materialinclude sugar caner bagasses, sugar beet pulp, corn stover, wood chips,sawdust and switchgrass.

The term “co-culture”, and variants thereof such as “co-cultivate”,refer to the presence of two or more types of cells in the samebioreactor. The two or more types of cells may both be microorganisms,such as microalgae, or may be a microalgal cell cultured with adifferent cell type. The culture conditions may be those that fostergrowth and/or propagation of the two or more cell types or those thatfacilitate growth and/or proliferation of one, or a subset, of the twoor more cells while maintaining cellular growth for the remainder.

“Combination Product” means a product that comprises at least twodistinct compositions intended for human administration through distinctroutes, such as a topical route and an oral route. In some embodimentsthe same active agent is contained in both the topical and oralcomponents of the combination product.

“Conditions favorable to cell division” means conditions in which cellsdivide at least once every 72 hours.

The term “cultivated”, and variants thereof, refer to the intentionalfostering of growth (increases in cell size, cellular contents, and/orcellular activity) and/or propagation (increases in cell numbers viamitosis) of one or more cells by use of intended culture conditions. Thecombination of both growth and propagation may be termed proliferation.The one or more cells may be those of a microorganism, such asmicroalgae. Examples of intended conditions include the use of a definedmedium (with known characteristics such as pH, ionic strength, andcarbon source), specified temperature, oxygen tension, carbon dioxidelevels, and growth in a bioreactor. The term does not refer to thegrowth or propagation of microorganisms in nature or otherwise withoutdirect human intervention.

“DHA” means Docosahexaenoic acid.

“Endopolysaccharide” means a polysaccharide that is retainedintracellularly.

“EPA” means eicosapentaenoic acid.

Cells or biomass that are “essentially free of red coloration” containeither no red color visible to the naked eye or a small amount of redcolor such that red is a minor color of the overall composition comparedto at least one other color such as yellow.

Cells or biomass that are “completely free of red coloration” contain nored color visible to the naked eye.

“Exogenous gene” refers to a nucleic acid transformed into a cell. Atransformed cell may be referred to as a recombinant cell, into whichadditional exogenous gene(s) may be introduced. The exogenous gene maybe from a different species (and so heterologous), or from the samespecies (and so homologous) relative to the cell being transformed. Inthe case of a homologous gene, it occupies a different location in thegenome of the cell relative to the endogenous copy of the gene. Theexogenous gene may be present in more than one copy in the cell. Theexogenous gene may be maintained in a cell as an insertion into thegenome or as an episomal molecule.

“Exogenously provided” describes a molecule provided to the culturemedia of a cell culture.

“Exopolysaccharide” means a polysaccharide that is secreted from a cellinto the extracellular environment.

As used herein, the terms “expression vector” or “expression construct”refer to a nucleic acid construct, generated recombinantly orsynthetically, with a series of specified nucleic acid elements thatpermit transcription of a particular nucleic acid in a host cell. Theexpression vector can be part of a plasmid, virus, or nucleic acidfragment. Typically, the expression vector includes a nucleic acid to betranscribed operably linked to a promoter.

“Filtrate” means the portion of a tangential flow filtration sample thathas passed through the filter.

“Fixed carbon source” means molecule(s) containing carbon that arepresent at ambient temperature and pressure in solid or liquid form.

“Glycopolymer” means a biologically produced molecule comprising atleast two monosaccharides. Examples of glycopolymers includeglycosylated proteins, polysaccharides, oligosaccharides, anddisaccharides.

“Homogenate” means cell biomass that has been disrupted. A homogenate isnot necessarily homogeneous.

As used herein, the term “lysate” refers to a solution containing thecontents of lysed cells.

As used herein, the term “lysis” refers to the breakage of the plasmamembrane and optionally the cell wall of a biological organismsufficient to release at least some intracellular content, often bymechanical, viral or osmotic mechanisms that compromise its integrity.

As used herein, the term “lysing” refers to disrupting the cellularmembrane and optionally the cell wall of a biological organism or cellsufficient to release at least some intracellular content.

A compound that can be “metabolized by cells” means a compound whoseelemental components are incorporated into products endogenouslyproduced by the cells. For example, a compound containing nitrogen thatcan be metabolized by cells is a compound containing at least onenitrogen atom per molecule that can be incorporated into anitrogen-containing, endogenously produced metabolite such as an aminoacid.

“Microalgae” means a eukarytotic microbial organism that contains achloroplast, and optionally that is capable of performingphotosynthesis, or a prokaryotic microbial organism capable ofperforming photosynthesis. Microalgae include obligate photoautotrophs,which cannot metabolize a fixed carbon source as energy, as well asheterotrophs, which can live solely off of a fixed carbon source.Microalgae can refer to unicellular organisms that separate from sistercells shortly after cell division, such as Chlamydomonas, and can alsorefer to microbes such as, for example, Volvox, which is a simplemulticellular photosynthetic microbe of two distinct cell types.“Microalgae” can also refer to cells such as Chlorella and Dunaliella.“Microalgae” also includes other microbial photosynthetic organisms thatexhibit cell-cell adhesion, such as Agmenellum, Anabaena, andPyrobotrys. “Microalgae” also includes obligate heterotrophicmicroorganisms that have lost the ability to perform photosynthesis,such as certain dinoflagellate algae species.

The terms “microorganism” and “microbe” are used interchangeably hereinto refer to microscopic unicellular organisms.

“Naturally produced” describes a compound that is produced by awild-type organism.

“Peptide” means a polypeptide of 50 or less amino acids. In somecontexts, a peptide is connected to a much larger protein as a fusionprotein and is referred to as a peptide to denote its independent domainas a part of the fusion protein.

“Photobioreactor” means a waterproof container, at least part of whichis at least partially transparent, allowing light to pass through, inwhich one or more microalgae cells are cultured. Photobioreactors may besealed, as in the instance of a polyethylene bag, or may be open to theenvironment, as in the instance of a pond.

“Polysaccharide material” is a composition that contains more than onespecies of polysaccharide, and optionally contaminants such as proteins,lipids, and nucleic acids, such as, for example, a microalgal cellhomogenate.

“Polysaccharide” means a compound or preparation containing one or moremolecules that contain at least two saccharide molecules covalentlylinked. A “polysaccharide”, “endopolysaccharide” or “exopolysaccharide”can be a preparation of polymer molecules that have similar or identicalrepeating units but different molecular weights within the population.

“Port”, in the context of a photobioreactor, means an opening in thephotobioreactor that allows influx or efflux of materials such as gases,liquids, and cells. Ports are usually connected to tubing leading toand/or from the photobioreactor.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

As used herein, the term “recombinant” when used with reference, e.g.,to a cell, or nucleic acid, protein, or vector, indicates that the cell,nucleic acid, protein or vector, has been modified by the introductionof an exogenous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as depicted above.

“Red microalgae” means unicellular algae that is of the list of classescomprising Bangiophyceae, Florideophyceae, Goniotrichales, or isotherwise a member of the Rhodophyta.

“Retentate” means the portion of a tangential flow filtration samplethat has not passed through the filter.

“Small molecule” means a molecule having a molecular weight of less than2000 daltons, in some instances less than 1000 daltons, and in stillother instances less than 500 daltons or less. Such molecules include,for example, heterocyclic compounds, carbocyclic compounds, sterols,amino acids, lipids, carotenoids and polyunsaturated fatty acids.

A molecule is “solvent available” when the molecule is isolated to thepoint at which it can be dissolved in a solvent, or sufficientlydispersed in suspension in the solvent such that it can be detected inthe solution or suspension. For example, a polysaccharide is “solventavailable” when it is sufficiently isolated from other materials, suchas those with which it is naturally associated, such that thepolysaccharide can be dissolved or suspended in an aqueous buffer anddetected in solution using a dimethylmethylene blue (DMMB) orphenol:sulfuric acid assay. In the case of a high molecular weightpolysaccharide containing hundreds or thousands of monosaccharides, partof the polysaccharide can be “solvent available” when it is on theoutermost layer of a cell wall while other parts of the samepolysaccharide molecule are not “solvent available” because they areburied within the cell wall. For example, in a culture of microalgae inwhich polysaccharide is present in the cell wall, there is little“solvent available” polysaccharide since most of the cell wallpolysaccharide is sequestered within the cell wall and not available tosolvent. However, when the cells are disrupted, e.g., by sonication, theamount of “solvent available” polysaccharide increases. The amount of“solvent accessible” polysaccharide before and after homogenization canbe compared by taking two aliquots of equal volume of cells from thesame culture, homogenizing one aliquot, and comparing the level ofpolysaccharide in solvent from the two aliquots using a DMMB assay. Theamount of solvent accessible polysaccharide in a homogenate of cells canalso be compared with that present in a quantity of cells of the sametype in a different culture needed to generate the same amount ofhomogenate.

“Substantially free of protein” means compositions that are preferablyof high purity and are substantially free of potentially harmfulcontaminants, including proteins (e.g., at least National Food (NF)grade, generally at least analytical grade, and more typically at leastpharmaceutical grade). Compositions are at least 80, at least 90, atleast 99 or at least 99.9% w/w pure of undesired contaminants such asproteins are substantially free of protein. To the extent that a givencompound must be synthesized prior to use, the resulting product istypically substantially free of any potentially toxic agents,particularly any endotoxins, which may be present during the synthesisor purification process. Compositions are usually made under GMPconditions. Compositions for parenteral administration are usuallysterile and substantially isotonic.

A “sucrose utilization gene” is a gene that, when expressed, aids theability of a cell to utilize sucrose as an energy source. Proteinsencoded by a sucrose utilization gene are referred to herein as “sucroseutilization enzymes” and include sucrose transporters, sucroseinvertases, and hexokinases such as glucokinases and fructokinases.

For sequence comparison to determine percent nucleotide or amino acididentity, typically one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat.'l. Acad. Sci. USA 85: 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

Another example algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (at the web addresswww.ncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra.). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. For identifying whether a nucleicacid or polypeptide is within the scope of the invention, the defaultparameters of the BLAST programs are suitable. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. TheTBLATN program (using protein sequence for nucleotide sequence) uses asdefaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix. (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

II. General

Polysaccharides form a heterogeneous group of polymers of differentlength and composition. They are constructed from monosaccharideresidues that are linked by glycosidic bonds. Glycosidic linkages may belocated between the C₁ (or C₂) of one sugar residue and the C₂, C₃, C₄,C₅ or C₆ of the second residue. A branched sugar results if more thantwo types of linkage are present in single monosaccharide molecule.

Monosaccharides are simple sugars with multiple hydroxyl groups. Basedon the number of carbons (e.g., 3, 4, 5, or 6) a monosaccharide is atriose, tetrose, pentose, or hexose. Pentoses and hexoses can cyclize,as the aldehyde or keto group reacts with a hydroxyl on one of thedistal carbons. Examples of monosaccharides are galactose, glucose, andrhamnose.

Polysaccharides are molecules comprising a plurality of monosaccharidescovalently linked to each other through glycosidic bonds.Polysaccharides consisting of a relatively small number ofmonosaccharide units, such as 10 or less, are sometimes referred to asoligosaccharides. The end of the polysaccharide with an anomeric carbon(C₁) that is not involved in a glycosidic bond is called the reducingend. A polysaccharide may consist of one monosaccharide type, known as ahomopolymer, or two or more types of monosaccharides, known as aheteropolymer. Examples of homopolysaccharides are cellulose, amylose,inulin, chitin, chitosan, amylopectin, glycogen, and pectin. Amylose isa glucose polymer with α(1→4) glycosidic linkages. Amylopectin is aglucose polymer with α(1→4) linkages and branches formed by α(1→6)linkages. Examples of heteropolysaccharides are glucomannan,galactoglucomannan, xyloglucan, 4-O-methylglucuronoxylan, arabinoxylan,and 4-O-Methylglucuronoarabinoxylan.

Polysaccharides can be structurally modified both enzymatically andchemically. Examples of modifications include sulfation,phosphorylation, methylation, O-acetylation, fatty acylation, aminoN-acetylation, N-sulfation, branching, and carboxyl lactonization.

Glycosaminoglycans are polysaccharides of repeating disaccharides.Within the disaccharides, the sugars tend to be modified, with acidicgroups, amino groups, sulfated hydroxyl and amino groups.Glycosaminoglycans tend to be negatively charged, because of theprevalence of acidic groups. Examples of glycosaminoglycans are heparin,chondroitin, and hyaluronic acid.

Polysaccharides are produced in eukaryotes mainly in the endoplasmicreticulum (ER) and Golgi apparatus. Polysaccharide biosynthesis enzymesare usually retained in the ER, and amino acid motifs imparting ERretention have been identified (Gene. 2000 Dec. 31; 261(2): 321-7).Polysaccharides are also produced by some prokaryotes, such as lacticacid bacteria.

Polysaccharides that are secreted from cells are known asexopolysaccharides. Many types of cell walls, in plants, algae, andbacteria, are composed of polysaccharides. The cell walls are formedthrough secretion of polysaccharides. Some species, including algae andbacteria, secrete polysaccharides that are released from the cells. Inother words, these molecules are not held in association with the cellsas are cell wall polysaccharides. Instead, these molecules are releasedfrom the cells. For example, cultures of some species of microalgaesecrete exopolysaccharides that are suspended in the culture media.

III. Methods of Producing Polysaccharides

A. Cell Culture Methods: Microalgae

Polysaccharides can be produced by culturing microalgae. Examples ofmicroalgae that can be cultured to produce polysaccharides are shown inTables 1A and 1B. Also listed in Table 1A are references that enable theskilled artisan to culture the microalgae species under conditionssufficient for polysaccharide production. Also listed in Table 1A arestrain numbers from various publicly available algae collections, aswell as strains published in journals that require public disseminationof reagents as a prerequisite for publication.

TABLE 1A Examples of microalgae, culture parameters, and polysaccharideproduction. Culture and polysaccharide Reported Strain Number/purification method Monosaccharide Species Source reference CompositionCulture conditions Porphyridium UTEX¹ 161 M. A. Guzman-Murillo andXylose, Glucose, Cultures obtained from various cruentum F. Ascencio.,Letters in Galactose, sources and were cultured in F/2 broth AppliedMicrobiology 2000, Glucoronic acid prepared with seawater filtered 30,473-478 through a 0.45 um Millipore filter or distilled water dependingon microalgae salt tolerance. Incubated at 25° C. in flasks andilluminated with white fluorescent lamps. Porphyridium UTEX 161 Fabregaset al., Antiviral Xylose, Glucose, Cultured in 80 ml glass tubes withcruentum Research 44(1999)-67-73 Galactose and aeration of 100 ml/minand 10% CO₂, Glucoronic acid for 10 s every ten minutes to maintainpH >7.6. Maintained at 22° in 12:12 Light/dark periodicity. Light at152.3 umol/m2/s. Salinity 3.5% (nutrient enriched as Fabregas, 1984modified in 4 mmol Nitrogen/L) Porphyridium sp. UTEX 637 Dvir, Brit. J.of Nutrition Xylose, Glucose Outdoor cultivation for 21 days in (2000),84, 469-476. and Galactose, artficial sea water in polyethylene [Review:S. Geresh Methyl hexoses, sleeves. See Jones (1963) and Cohen BiosourceTechnology 38 Mannose, & Malis Arad, 1989) (1991) 195-201]-Huleihel,Rhamnose 2003, Applied Spectoscopy, v57, No. 4 2003 Porphyridium SAG²111.79 Talyshinsky, Marina Cancer xylose, glucose, see Dubinsky et al.Plant Physio. And aerugineum Cell Int'l 2002, 2; Review: galactose,methyl Biochem. (192) 30: 409-414. Pursuant S. Geresh Biosource hexosesto Ramus_1972--> Axenic culutres Technology 38 (1991) 195-201] are grownin MCYII liquid medium at 1 See Ramus_1972 25° C. and illuminated withCool White fluorescent tubes on a 16:8 hr light dark cycle. Cells keptin suspension by agitation on a gyrorotary shaker or by a stream offiltered air. Porphyridium strain 1380-1a Schmitt D., Water Researchunknown See cited reference purpurpeum Volume 35, Issue 3, March 2001,Pages 779-785, Bioprocess Biosyst Eng. 2002 Apr; 25(1): 35-42. Epub 2002Mar 6 Chaetoceros sp. USCE³ M. A. Guzman-Murillo and unknown See citedreference F. Ascencio., Letters in Applied Microbiology 2000, 30,473-478 Chlorella USCE M. A. Guzman-Murillo and unknown See citedreference autotropica F. Ascencio., Letters in Applied Microbiology2000, 30, 473-478 Chlorella UTEX 580 Fabregas et al., Antiviral unknownCultured in 80 ml glass tubes with autotropica Research 44(1999)-67-73aeration of 100 ml/min and 10% CO2, for 10 s every ten minutes tomaintain pH >7.6. Maintained at 22° in 12:12 Light/dark periodicity.Light at 152.3 umol/m2/s. Salinity 3.5% (nutrient enriched as Fabregas,1984) Chlorella UTEX LB2074 M. A. Guzman-Murillo and Un known Culturesobtained from various capsulata F. Ascencio., Letters in sources andwere cultured in F/2 broth Applied Microbiology 2000, prepared withseawater filtered 30, 473-478 (species is through a 0.45 um Milliporefilter or a.k.a. Schizochlamydella distilled water depending oncapsulata) microalgae salt tolerance. Incubated at 25° C. in flasks andilluminated with white fluorescent lamps. Chlorella GGMCC⁴ S. Guzman,Phytotherapy glucose, Grown in 10 L of membrane filtered stigmatophoraRscrh (2003) 17: 665-670 glucuronic acid, (0.24 um) seawater andsterilized at xylose, 120° for 30 min and enriched with Erdribose/fucose Schreiber medium. Cultures maintained at 18 +/− 1° C.under constant 1% CO₂ bubbling. Dunalliela DCCBC⁵ Fabregas et al.,Antiviral unknown Cultured in 80 ml glass tubes with tertiolectaResearch 44(1999)-67-73 aeration of 100 ml/min and 10% CO2, for 10 severy ten minutes to maintain pH >7.6. Maintained at 22° in 12:12Light/dark periodicity. Light at 152.3 umol/m2/s. Salinity 3.5%(nutrient enriched as Fabregas, 1984) Dunalliela DCCBC Fabregas et al.,Antiviral unknown Cultured in 80 ml glass tubes with bardawil Research44(1999)-67-73 aeration of 100 ml/min and 10% CO2, for 10 s every tenminutes to maintain pH >7.6. Maintained at 22° in 12:12 Light/darkperiodicity. Light at 152.3 umol/m²/s. Salinity 3.5% (nutrient enrichedas Fabregas, 1984) Isochrysis galbana HCTMS⁶ M. A. Guzman-Murillo andunknown Cultures obtained from various var. tahitiana F. Ascencio.,Letters in sources and were cultured in F/2 broth Applied Microbiology2000, prepared with seawater filtered 30, 473-478 through a 0.45 ummillipore filter or distilled water depending on microalgae salttolerance. Incubated at 25° C. in flasks and illuminated with whitefluorescent lamps. Isochrysis galbana UTEX LB 987 Fabregas et al.,Antiviral unknown Cultured in 80 ml glass tubes with var. Tiso Research44(1999)-67-73 aeration of 100 ml/min and 10% CO2, for 10 s every tenminutes to maintain pH >7.6. Maintained at 22° in 12:12 Light/darkperiodicity. Light at 152.3 umol/m²/s. Salinity 3.5% (nutrient enrichedas Fabregas, 1984) Isochrysis sp. CCMP⁷ M. A. Guzman-Murillo and unknownCultures obtained from various F. Ascencio., Letters in sources and werecultured in F/2 broth Applied Microbiology 2000, prepared with seawaterfiltered 30, 473-478 through a 0.45 um Millipore filter or distilledwater depending on microalgae salt tolerance. Incubated at 25° C. inflasks and illuminated with white fluorescent lamps. Phaeodactylum UTEX642, 646, M. A M. A. Guzman-Murillo unknown Cultures obtained fromvarious tricornutum 2089 and F. Ascencio., Letters in sources and werecultured in F/2 broth Applied Microbiology 2000, prepared with seawaterfiltered 30, 473-478 through a 0.45 um Millipore filter or distilledwater depending on microalgae salt tolerance. Incubated at 25° C. inflasks and illuminated with white fluorescent lamps. Phaeodactylum GGMCCS. Guzman, Phytotherapy glucose, Grown in 10 L of membrane filteredtricornutum Rscrh (2003) 17: 665-670 glucuronic acid, (0.24 um) seawaterand sterilized at and mannose 120° for 30 min and enriched with ErdSchreiber medium. Cultures maintained at 18 +/− 1° C. under constant 1%CO2 bubbling. Tetraselmis sp. CCMP 1634- M. A. Guzman-Murillo andunknown Cultures obtained from various 1640; UTEX F. Ascencio., Lettersin sources and were cultured in F/2 broth 2767 Applied Microbiology2000, prepared with seawater filtered 30, 473-478 through a 0.45 umMillipore filter or distilled water depending on microalgae salttolerance. Incubated at 25° C. in flasks and illuminated with whitefluorescent lamps. Botrycoccus UTEX 572 and M. A. Guzman-Murillo andunknown Cultures obtained from various braunii 2441 F. Ascencio.,Letters in sources and were cultured in F/2 broth Applied Microbiology2000, prepared with seawater filtered 30, 473-478 through a 0.45 umMillipore filter or distilled water depending on microalgae salttolerance. Incubated at 25° C. in flasks and illuminated with whitefluorescent lamps. Cholorococcum UTEX 105 M. A. Guzman-Murillo andunknown Cultures obtained from various F. Ascencio., Letters in sourcesand were cultured in F/2 broth Applied Microbiology 2000, prepared withseawater filtered 30, 473-478 through a 0.45 um Millipore filter ordistilled water depending on microalgae salt tolerance. Incubated at 25°C. in flasks and illuminated with white fluorescent lamps. HormotilopsisUTEX 104 M. A. Guzman-Murillo and unknown Cultures obtained from variousgelatinosa F. Ascencio., Letters in sources and were cultured in F/2broth Applied Microbiology 2000, prepared with seawater filtered 30,473-478 through a 0.45 um Millipore filter or distilled water dependingon microalgae salt tolerance. Incubated at 25° C. in flasks andilluminated with white fluorescent lamps. Neochloris UTEX 1185 M. A.Guzman-Murillo and unknown Cultures obtained from various oleoabundansF. Ascencio., Letters in sources and were cultured in F/2 broth AppliedMicrobiology 2000, prepared with seawater filtered 30, 473-478 through a0.45 um Millipore filter or distilled water depending on microalgae salttolerance. Incubated at 25° C. in flasks and illuminated with whitefluorescent lamps. Ochromonas UTEX L1298 M. A. Guzman-Murillo andunknown Cultures obtained from various Danica F. Ascencio., Letters insources and were cultured in F/2 broth Applied Microbiology 2000,prepared with seawater filtered 30, 473-478 through a 0.45 um Milliporefilter or distilled water depending on microalgae salt tolerance.Incubated at 25° C. in flasks and illuminated with white fluorescentlamps. Gyrodinium KG03; KGO9; Yim, Joung Han et. Al., J. ofHomopolysaccharide Isolated from seawater collected from impudicum KGJO1Microbiol December 2004, 305-14; of galactose w/ red-tide bloom inKorean coastal Yim, J. H. (2000) Ph.D. 2.96% uronic acid water.Maintained in f/2 medium at Dissertations, University of 22° undercircadian light at Kyung Hee, Seoul 100 uE/m2/sec: dark cycle of 14 h:10 h for 19 days. Selected with neomycin and/or cephalosporin 20 ug/mlEllipsoidon sp. See cited Fabregas et al., Antiviral unknown Cultured in80 ml glass tubes with references Research 44(1999)-67-73; aeration of100 ml/min and 10% CO2, Lewin, R. A. Cheng, L., for 10 s every tenminutes to maintain 1989. Phycologya 28, 96-108 pH >7.6. Maintained at22° in 12:12 Light/dark periodicity. Light at 152.3 umol/m2/s. Salinity3.5% (nutrient enriched as Fabregas, 1984) Rhodella UTEX 2320Talyshinsky, Marina Cancer unknown See Dubinsky O. et al. Composition ofreticulata Cell Int'l 2002, 2 Cell wall polysaccharide produced byunicellular red algae Rhodella reticulata. 1992 Plant Physiology andbiochemistry 30: 409-414 Rhodella UTEX LB 2506 Evans, LV., et al. J.Cell Sci Galactose, xylose, Grown in either SWM3 medium or maculata 16,1-21(1974); EVANS, L. V. glucuronic acid ASP12, MgCl2 supplement. 100mls (1970). Br. phycol.J. 5, 1-13. in 250 mls volumetric Erlenmeyerflask with gentle shaking and 4000lx Northern Light fluorescent lightfor 16 hours. Gymnodinium sp. Oku-1 Sogawa, K., et al., Life unknown Seecited reference Sciences, Vol. 66, No. 16, pp. PL 227-231 (2000) ANDUmermura, Ken: Biochemical Pharmacology 66 (2003) 481-487 Spirilinaplatensis UTEX LB 1926 Kaji, T et. Al., Life Sci 2002 Na-Sp contains Seecited reference Mar 8; 70(16): 1841-8 two disaccharide Schaeffer andKrylov (2000) repeats: Review-Ectoxicology and Aldobiuronic acidEnvironmental Safety. 45, and Acofriose + 208-227. other minorsaccharides and sodium ion Cochlodinuium Oku-2 Hasui., et. Al., Int. J.Bio. mannose, Precultures grown in 500 ml conicals polykrikoidesMacromol. Volume 17 No. galactose, glucose containing 300 mls ESM (?) at21.5° C. 5 1995. and uronic acid for 14 days in continuous light (3500lux) in growth cabinet) and then transferred to 5 liter conical flaskcontaining 3 liters of ESM. Grown 50 days and then filtered thruwortmann GFF filter. Nostoc muscorum PCC⁸ 7413, Sangar, VK AppliedMicro. unknown Growth in nitrogen fixing conditions 7936, 8113 (1972) &A. M. Burja et al in BG-11 medium in aerated cultures Tetrahydron 57(2001) 937-9377; maintained in log phase for several Otero A., J months.250 mL culture media that Biotechnol. 2003 Apr were disposed in atemperature 24; 102(2): 143-52 controlled incubator and continuouslyilluminated with 70 umol photon m−2 s−1 at 30° C. Cyanospira See citedA. M. Burja et al. unknown See cited references capsulata referencesTetrahydron 57 (2001) 937-9377 & Garozzo, D., Carbohydrate Res. 1998 307113-124; Ascensio, F., Folia Microbiol (Praha). 2004; 49(1): 64-70.,Cesaro, A., et al., Int J Biol Macromol. 1990 Apr; 12(2): 79-84Cyanothece sp. ATCC 51142 Ascensio F., Folia Microbiol unknownMaintained at 27° C. in ASN III (Praha). 2004; 49(1): 64-70. medium withlight/dark cycle of 16/8 h under fluorescent light of 3,000 lux lightintensity. In Phillips each of 15 strains were grownphotoautotrophically in enriched seawater medium. When required theamount of NaNO3 was reduced from 1.5 to 0.35 g/L. Strains axenicallygrown in an atmosphere of 95% air and 5% CO2 for 8 days under continuousillumination. with mean photon flux of 30 umol photon/m2/s for the first3 days of growth and 80 umol photon/m/s Chlorella UTEX 343; Cheng_2004Journal of unknown See cited reference pyrenoidosa UTEX 1806 MedicinalFood 7(2) 146-152 Phaeodactylum CCAP 1052/1A Fabregas et al., Antiviralunknown Cultured in 80 ml glass tubes with tricornutum Research44(1999)-67-73 aeration of 100 ml/min and 10% CO2, for 10 s every tenminutes to maintain pH >7.6. Maintained at 22° in 12:12 Light/darkperiodicity. Light at 152.3 umol/m2/s. Salinity 3.5% (nutrient enrichedas Fabregas, 1984) Chlorella USCE M. A. Guzman-Murillo and unknown Seecited reference autotropica F. Ascencio., Letters in AppliedMicrobiology 2000, 30, 473-478 Chlorella sp. CCM M. A. Guzman-Murilloand unknown See cited reference F. Ascencio., Letters in AppliedMicrobiology 2000, 30, 473-478 Dunalliela USCE M. A. Guzman-Murillo andunknown See cited reference tertiolecta F. Ascencio., Letters in AppliedMicrobiology 2000, 30, 473-478 Isochrysis UTEX LB 987 Fabregas et al,Antiviral unknown Cultured in 80 ml glass tubes with galabana Research44(1999)-67-73 aeration of 100 ml/min and 10% CO₂, for 10 s every tenminutes to maintain pH >7.6. Maintained at 22° in 12:12 Light/darkperiodicity. Light at 152.3 umol/m2/s. Salinity 3.5% (nutrient enrichedas Fabregas, 1984) Tetraselmis CCAP 66/1A-D Fabregas et al., Antiviralunknown Cultured in 80 ml glass tubes with tetrathele Research44(1999)-67-73 aeration of 100 ml/min and 10% CO₂, for 10 s every tenminutes to maintain pH >7.6. Maintained at 22° in 12:12 Light/darkperiodicity. Light at 152.3 umol/m2/s. Salinity 3.5% (nutrient enrichedas Fabregas, 1984) Tetraselmis UTEX LB 2286 M. A. Guzman-Murillo andunknown See cited reference suecica F. Ascencio., Letters in AppliedMicrobiology 2000, 30, 473-478 Tetraselmis CCAP 66/4 Fabregas et al.,Antiviral unknown Cultured in 80 ml glass tubes with suecica Research44(1999)-67-73 aeration of 100 ml/min and 10% CO₂, and Otero andFabregas- for 10 s every ten minutes to maintain Aquaculture 159 (1997)pH >7.6. Maintained at 22° in 12:12 111-123. Light/dark periodicity.Light at 152.3 umol/m2/s. Salinity 3.5% (nutrient enriched as Fabregas,1984) Botrycoccus UTEX 2629 M. A. Guzman-Murillo and unknown See citedreference sudeticus F. Ascencio., Letters in Applied Microbiology 2000,30, 473-478 Chlamydomonas UTEX 729 Moore and Tisher Science. unknown Seecited reference mexicana 1964 Aug. 7; 145: 586-7. Dysmorphococcus UTEXLB 65 M. A. Guzman-Murillo and unknown See cited reference globosus F.Ascencio., Letters in Applied Microbiology 2000, 30, 473-478 RhodellaUTEX LB 2320 S. Geresh et al., J Biochem. unknown See cited referencereticulata Biophys. Methods 50 (2002) 179-187 [Review: S. GereshBiosource Technology 38 (1991) 195-201] Anabena ATCC 29414 Sangar, VKAppl Microbiol. In Vegative wall See cited reference cylindrica 1972Nov; 24(5): 732-4 where only 18% is carbohydrate- Glucose [35%], mannose[50%], galactose, xylose, and fucose. In heterocyst wall where 73% iscarbohydrate- Glucose 73% and Mannose is 21% with some galactose andxylose Anabena flosaquae A37; JM Moore, BG [1965] Can J. Glucose and Seecited reference and APPLIED Kingsbury Microbiol. Dec; 11(6): 877-85mannose ENVIRONMENTAL Laboratory, MICROBIOLOGY, April 1978, 718-723)Cornell University Palmella mucosa See cited Sangar, VK Appl Microbiol.unknown See cited reference references 1972 Nov; 24(5): 732-4; LewinRA., (1956) Can J Microbiol. 2: 665-672; Arch Mikrobiol. 1964 Aug 17;49: 158-66 Anacystis PCC 6301 Sangar, VK Appl Microbiol. Glucose, Seecited reference nidulans 1972 Nov; 24(5): 732-4 galactose, mannosePhormidium 94a See cited Vicente-Garcia V. et al., Galactose, Cultivatedin 2 L BG-11 medium at reference Biotechnol Bioeng. 2004 Mannose, 28° C.Acetone was added to Feb 5; 85(3): 306-10 Galacturonic acid, precipitateexopolysaccharide. Arabinose, and Ribose Anabaenaopsis 1402/1⁹ David KA,Fay P. Appl unknown See cited reference circularis Environ Microbiol.1977 Dec; 34(6): 640-6 Aphanocapsa MN-11 Sudo H., et al. CurrentRhamnose; Cultured aerobically for 20 days in halophtia MicrcobiologyVol. 30 mannose; fucose; seawater-based medium, with 8% (1995),pp.219-222 galactose; xylose; NaCl, and 40 mg/L NaHPO4. Nitrate glucoseIn ratio of: changed the Exopolysaccharide 15:53:3:3:25 content. Highestcell density was obtained from culture supplemented with 100 mg/l NaNO₃.Phosphorous (40 mg/L) could be added to control the biomass andexopolysaccharide concentration. Aphanocapsa sp See reference DePhilippis R et al., Sci unknown Incubated at 20 and 28° C. with TotalEnviron. 2005 Nov 2; artificial light at a photon flux of 5-20 umol m⁻²s⁻¹. Cylindrotheca sp See reference De Philippis R et al., SciGlucuronic acid, Stock enriched cultures incubated at Total Environ.2005 Nov 2; Galacturonic acid, 20 and 28° C. with artificial light at aGlucose, photon flux of 5-20 umol m−2 s−1. Mannose, Exopolysaccharideproduction done in Arabinose, glass tubes containing 100 mL cultureFructose and at 28° C. with continuous illumination Rhamnose at photondensity of 5-10 uE m−2 s−1. Navicula sp See reference De Philippis R etal., Sci Glucuronic acid, Incubated at 20 and 28° C. with Total Environ.2005 Nov 2; Galacturonic acid, artificial light at a photon flux of 5-20umol Glucose, m−2 s−1. EPS production done in Mannose, glass tubescontaining 100 mL culture Arabinose, at 28° C. with continuousillumination Fructose and at photon density of 5-10 uE m−2 s−1. RhamnoseGloeocapsa sp See reference De Philippis R et al., Sci unknown Incubatedat 20 and 28° C. with artifical Total Environ. 2005 Nov 2; light at aphoton flux of 5-20 umol m−2 s−1. Gloeocapsa See cited J Appl Microbiol.unknown See cited references alpicola references 2005; 98(1): 114-20;Photochem Photobiol. 1982 Mar; 35(3): 359-64; J Gen Microbiol. 1977 Jan;98(1): 277-80; Arch Microbiol. 1976 Feb; 107(1): 93-7; FEMS MicrobiolLett. 2002 Sep 10; 214(2): 229-33 Phaeocystis See cited Toxicology. 2004Jul unknown See cited references pouchetii references 1; 199(2-3):207-17; Toxicon. 2003 Jun; 41(7): 803-12; Protist. 2002 Sep; 153(3):275-82; J Virol. 2005 Jul; 79(14): 9236-43; J Bacteriol. 1961 Jul;82(1): 72-9 Leptolyngbya sp See reference De Philippis R et al., Sciunknown Incubated at 20 and 28° C. with Total Environ. 2005 Nov 2;artificial light at a photon flux of 5-20 umol m−2 s−1. Symploca sp. Seereference De Philippis R et al., Sci unknown Incubated at 20 and 28° C.with Total Environ. 2005 Nov 2; artificial light at a photon flux of5-20 umol m−2 s−1. Synechocystis PCC 6714/6803 Jurgens UJ, Weckesser J.J Glucoseamine, Photoautotrophically grown in BG-11 Bacteriol. 1986mannosamine, medium, pH 7.5 at 25° C. Mass Nov; 168(2): 568-73galactosamine, cultures prepared in a 12 liter mannose and fermentor andgassed by air and glucose carbon dioxide at flow rates of 250 and 2.5liters/h, with illumination from white fluorescent lamps at a constantlight intensity of 5,000 lux. Stauroneis See reference Lind, JL (1997)Planta unknown See cited reference decipiens 203: 213-221 AchnanthesIndiana Holdsworth, RH., Cell Biol. unknown See cited reference brevipesUniversity 1968 Jun; 37(3): 831-7; Acta Culture Cient Venez. 2002;53(1): 7-14.; Collection J. Phycol 36 pp. 882-890 (2000) AchnanthesStrain 330 from Wang, Y., et al., Plant unknown See cited referencelongipes National Institute Physiol. 1997 for Apr; 113(4): 1071-1080.Environmental Studies

Other nonlimiting examples of microalgae that can be used in accordancewith the present invention can be found in Table 1B.

TABLE 1B Examples of microalgae. Achnanthes orientalis, Agmenellum,Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis linea,Amphora coffeiformis punctata, Amphora coffeiformis taylori, Amphoracoffeiformis tenuis, Amphora delicatissima, Amphora delicatissimacapitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmusfalcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii,Botryococcus sudeticus, Bracteococcus minor, Bracteococcusmedionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri,Chaetoceros muelleri subsalsum, Chaetoceros sp., Chlorella anitrata,Chlorella Antarctica, Chlorella aureoviridis, Chlorella candida,Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea,Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata,Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var.actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri,Chlorella lobophora (strain SAG 37.88), Chlorella luteoviridis,Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var.lutescens, Chlorella miniata, Chlorella minutissima, Chlorellamutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva,Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides(including any of UTEX strains 1806, 411, 264, 256, 255, 250, 249, 31,29, 25), Chlorella protothecoides var. acidicola, Chlorella regularis,Chlorella regularis var. minima, Chlorella regularis var. umbricata,Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophilavar. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorellasorokiniana, Chlorella sp., Chlorella sphaerica, Chlorellastigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorellavulgaris f. tertia, Chlorella vulgaris var. autotrophica, Chlorellavulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorellavulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f.viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorellatrebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcumsp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp.,Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotellameneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil,Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime,Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliellaprimolecta, Dunaliella salina, Dunaliella terricola, Dunaliellatertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaeraviridis, Eremosphaera sp., Ellipsoidon sp., Euglena, Franceia sp.,Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnionsp., Hymenomonas sp., Isochrysis aff. galbana, Isochrysis galbana,Lepocinclis, Micractinium, Micractinium (UTEX LB 2614), Monoraphidiumminutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina,Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Naviculapseudotenelloides, Navicula pelliculosa, Navicula saprophila, Naviculasp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschiaalexandrina, Nitzschia communis, Nitzschia dissipata, Nitzschiafrustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschiaintermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusillaelliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular,Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla,Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoriasubbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp.,Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae,Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii,Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis,Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp.,Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmusarmatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcussp., Synechococcus sp., Tetraedron, Tetraselmis sp., Tetraselmissuecica, Thalassiosira weissflogii, and Viridiella fridericiana

Microalgae are preferably cultured in liquid media for polysaccharideproduction. Culture condition parameters can be manipulated to optimizetotal polysaccharide production as well as to alter the structure ofpolysaccharides produced by microalgae.

Microalgal culture media usually contains components such as a fixednitrogen source, trace elements, a buffer for pH maintenance, andphosphate. Other components can include a fixed carbon source such asacetate or glucose, and salts such as sodium chloride, particularly forseawater microalgae. Examples of trace elements include zinc, boron,cobalt, copper, manganese, and molybdenum in, for example, therespective forms of ZnCl₂, H₃BO₃, CoCl₂.6H₂O, CuCl₂.2H₂O, MnCl₂.4H₂O and(NH₄)₆Mo₇O₂₄.4H₂O.

Some microalgae species can grow by utilizing a fixed carbon source suchas glucose or acetate. Such microalgae can be cultured in bioreactorsthat do not allow light to enter. Alternatively, such microalgae canalso be cultured in photobioreactors that contain the fixed carbonsource and allow light to strike the cells. Such growth is known asheterotrophic growth. Any strain of microalgae, including those listedin Tables 1A and 1B, can be cultured in the presence of any one or morefixed carbon source including those listed in Tables 2 and 3.

TABLE 2 Examples of fixed-carbon sources for culturing microalgae.2,3-Butanediol; 2-Aminoethanol; 2′-Deoxy Adenosine; 3-Methyl Glucose;Acetic Acid; Adenosine; Adenosine-5′-Monophosphate; Adonitol; Amygdalin;Arbutin; Bromosuccinic Acid; Cis-Aconitic Acid; Citric Acid;D,L-Carnitine; D,L-Lactic Acid; D,L-α-Glycerol Phosphate; D-Alanine;D-Arabitol; D-Cellobiose; Dextrin; D-Fructose; D-Fructose-6- Phosphate;D-Galactonic Acid Lactone; D-Galactose; D-Galacturonic Acid; D-GluconicAcid; D-Glucosaminic Acid; D-Glucose; D-Glucose-6-Phosphate;D-Glucuronic Acid; D- Lactic Acid Methyl Ester; D-L-α-GlycerolPhosphate; D-Malic Acid; D-Mannitol; D- Mannose; D-Melezitose;D-Melibiose; D-Psicose; D-Raffinose; D-Ribose; D-Saccharic Acid;D-Serine; D-Sorbitol; D-Tagatose; D-Trehalose; D-Xylose; Formic Acid;Gentiobiose; Glucuronamide; Glycerol; Glycogen; Glycyl-LAspartic Acid;Glycyl- LGlutamic Acid; Hydroxy-LProline; i-Erythritol; Inosine; Inulin;Itaconic Acid; Lactamide; Lactulose; L-Alaninamide; L-Alanine;L-Alanylglycine; L-Alanyl-Glycine; L-Arabinose; L-Asparagine; L-AsparticAcid; L-Fucose; L-Glutamic Acid; L-Histidine; L-Lactic Acid; L-Leucine;L-Malic Acid; L-Ornithine; LPhenylalanine; L-Proline; L-PyroglutamicAcid; L-Rhamnose; L-Serine; L-Threonine; Malonic Acid; Maltose;Maltotriose; Mannan; m- Inositol; N-Acetyl-DGalactosamine;N-Acetyl-DGlucosamine; N-Acetyl-LGlutamic Acid; N-Acetyl-β-DMannosamine;Palatinose; Phenyethylamine; p-Hydroxy-Phenylacetic Acid; PropionicAcid; Putrescine; Pyruvic Acid; Pyruvic Acid Methyl Ester; Quinic Acid;Salicin; Sebacic Acid; Sedoheptulosan; Stachyose; Succinamic Acid;Succinic Acid; Succinic Acid Mono-Methyl-Ester; Sucrose; Thymidine;Thymidine-5′-Monophosphate; Turanose; Tween 40; Tween 80; Uridine;Uridine-5′-Monophosphate; Urocanic Acid; Water; Xylitol; α-Cyclodextrin; α-D-Glucose; α-D-Glucose-1-Phosphate; α-D-Lactose;α-Hydroxybutyric Acid; α-Keto Butyric Acid; α-Keto Glutaric Acid; α-KetoValeric Acid; α-Ketoglutaric Acid; α-Ketovaleric Acid;α-Methyl-DGalactoside; α-Methyl-DGlucoside; α-Methyl- DMannoside;β-Cyclodextrin; β-Hydroxybutyric Acid; β-Methyl-DGalactoside; β-Methyl-D-Glucoside; γ-Amino Butyric Acid; and γ-Hydroxybutyric Acid

TABLE 3 Examples of fixed-carbon sources for culturing microalgae.(2-amino-3,4-dihydroxy-5-hydroxymethyl-1-cyclohexyl)glucopyranoside;(3,4- disinapoyl)fructofuranosyl-(6-sinapoyl)glucopyranoside;(3-sinapoyl)fructofuranosyl-(6- sinapoyl)glucopyranoside;1,10-di-O-(2-acetamido-2-deoxyglucopyranosyl)-2-azi-1,10- decanediol;1,3-mannosylmannose; 1,6-anhydrolactose; 1,6-anhydrolactose hexaacetate;1,6-dichlorosucrose; 1-chlorosucrose; 1-desoxy-1-glycinomaltose;1-O-alpha-2-acetamido- 2-deoxygalactopyranosyl-inositol;1-O-methyl-di-N-trifluoroacetyl-beta-chitobioside; 1- propyl-4-O-betagalactopyranosyl-alpha galactopyranoside; 2-(acetylamino)-4-O-(2-(acetylamino)-2-deoxy-4-O-sulfogalactopyranosyl)-2-deoxyglucose;2-(trimethylsilyl)ethyl lactoside; 2,1′,3′,4′,6′-penta-O-acetylsucrose;2,2′-O-(2,2′-diacetamido-2,3,2′,3′-tetradeoxy-6,6′-di-O-(2-tetradecylhexadecanoyl)-alpha,alpha′-trehalose-3,3′-diyl)bis(N-lactoyl-alanyl-isoglutamine); 2,3,6,2′,3′,4′,6′-hepta-O-acetylcellobiose;2,3′-anhydrosucrose; 2,3-di-O-phytanyl-1-O-(mannopyranosyl-(2-sulfate)-(1-2)-glucopyranosyl)-sn-glycerol;2,3- epoxypropyl O-galactopyranosyl(1-6)galactopyranoside;2,3-isoprolylideneerthrofuranosyl 2,3-O-isopropylideneerythrofuranoside;2′,4′-dinitrophenyl 2-deoxy-2-fluoro-beta- xylobioside;2,5-anhydromannitol iduronate; 2,6-sialyllactose;2-acetamido-2,4-dideoxy-4- fluoro-3-O-galactopyranosylglucopyranose;2-acetamido-2-deoxy-3-O-(gluco-4- enepyranosyluronic acid)glucose;2-acetamido-2-deoxy-3-O-rhamnopyranosylglucose; 2-acetamido-2-deoxy-6-O-beta galactopyranosylgalactopyranose;2-acetamido-2- deoxyglucosylgalactitol;2-acetamido-3-O-(3-acetamido-3,6-dideoxy-beta-glucopyranosyl)-2-deoxy-galactopyranose; 2-amino-6-O-(2-amino-2-deoxy-glucopyranosyl)-2-deoxyglucose; 2-azido-2-deoxymannopyranosyl-(1,4)-rhamnopyranose;2-deoxy-6-O-(2,3-dideoxy-4,6-O-isopropylidene-2,3-(N-tosylepimino)mannopyranosyl)-4,5-O-isopropylidene-1,3-di-N-tosylstreptamine; 2-deoxymaltose;2-iodobenzyl-1- thiocellobioside;2-N-(4-benzoyl)benzoyl-1,3-bis(mannos-4-yloxy)-2-propylamine; 2-nitrophenyl-2-acetamido-2-deoxy-6-O-beta galactopyranosyl-alphagalactopyranoside; 2-O- (glucopyranosyluronic acid)xylose;2-O-glucopyranosylribitol-1-phosphate; 2-O-glucopyranosylribitol-4′-phosphate;2-O-rhamnopyranosyl-rhamnopyranosyl-3-hydroxyldecanoyl-3-hydroxydecanoate; 2-O-talopyranosylmannopyranoside;2- thiokojibiose; 2-thiosophorose; 3,3′-neotrehalosadiamine; 3,6,3′,6′-dianhydro(galactopyranosylgalactopyranoside);3,6-di-O-methyl-beta-glucopyranosyl-(1-4)-2,3-di-O-methyl-alpha-rhamnopyranose;3-amino-3-deoxyaltropyranosyl-3-amino-3- deoxyaltropyranoside;3-deoxy-3-fluorosucrose; 3-deoxy-5-O-rhamnopyranosyl-2-octulopyranosonate; 3-deoxyoctulosonicacid-(alpha-2-4)-3-deoxyoctulosonic acid; 3- deoxysucrose;3-ketolactose; 3-ketosucrose; 3-ketotrehalose; 3-methyllactose; 3-O-(2-acetamido-6-O-(N-acetylneuraminyl)-2-deoxygalactosyl)serine;3-O-(glucopyranosyluronic acid)galactopyranose;3-O-beta-glucuronosylgalactose; 3-O-fucopyranosyl-2-acetamido-2-deoxyglucopyranose;3′-O-galactopyranosyl-1-4-O-galactopyranosylcytarabine; 3-O-galactosylarabinose; 3-O-talopyranosylmannopyranoside; 3-trehalosamine;4-(trifluoroacetamido)phenyl-2-acetamido-2-deoxy-4-O-beta-mannopyranosyl-beta-glucopyranoside;4,4′,6,6′-tetrachloro-4,4′,6,6′-tetradeoxygalactotrehalose; 4,6,4′,6′-dianhydro(galactopyranosylgalactopyranoside); 4,6-dideoxysucrose;4,6-O-(1-ethoxy-2- propenylidene)sucrose hexaacetate;4-chloro-4-deoxy-alpha-galactopyranosyl 3,4-anhydro-1,6-dichloro-1,6-dideoxy-beta-lyxo hexulofuranoside;4-glucopyranosylmannose; 4- methylumbelliferylcellobioside;4-nitrophenyl 2-fucopyranosyl-fucopyranoside; 4- nitrophenyl2-O-alpha-D-galactopyranosyl-alpha-D-mannopyranoside; 4-nitrophenyl 2-O-alpha-D-glucopyranosyl-alpha-D-mannopyranoside; 4-nitrophenyl2-O-alpha-D- mannopyranosyl-alpha-D-mannopyranoside; 4-nitrophenyl6-O-alpha-D-mannopyranosyl- alpha-D-mannopyranoside;4-nitrophenyl-2-acetamido-2-deoxy-6-O-beta-D-galactopyranosyl-beta-D-glucopyranoside; 4-O-(2-acetamido-2-deoxy-beta-glucopyranosyl)ribitol;4-O-(2-amino-2-deoxy-alpha-glucopyranosyl)-3-deoxy-manno-2- octulosonicacid; 4-O-(glucopyranosyluronic acid)xylose; 4-O-acetyl-alpha-N-acetylneuraminyl-(2-3)-lactose;4-O-alpha-D-galactopyranosyl-D-galactose; 4-O-galactopyranosyl-3,6-anhydrogalactose dimethylacetal;4-O-galactopyranosylxylose; 4-O-mannopyranosyl-2-acetamido-2-deoxyglucose; 4-thioxylobiose;4-trehalosamine; 4- trifluoroacetamidophenyl2-acetamido-4-O-(2-acetamido-2-deoxyglucopyranosyl)-2-deoxymannopyranosiduronic acid; 5-bromoindoxyl-beta-cellobioside; 5′-O-(fructofuranosyl-2-1-fructofuranosyl)pyridoxine;5-O-beta-galactofuranosyl- galactofuranose; 6 beta-galactinol;6(2)-thiopanose; 6,6′-di-O-corynomycoloyl-alpha-mannopyranosyl-alpha-mannopyranoside;6,6-di-O-maltosyl-beta-cyclodextrin; 6,6′-di-O-mycoloyl-alpha-mannopyranosyl-alpha-mannopyranoside;6-chloro-6-deoxysucrose; 6- deoxy-6-fluorosucrose;6-deoxy-alpha-gluco-pyranosiduronic acid; 6-deoxy-gluco- heptopyranosyl6-deoxy-gluco-heptopyranoside; 6-deoxysucrose; 6-O-decanoyl-3,4-di-O-isobutyrylsucrose; 6-O-galactopyranosyl-2-acetamido-2-deoxygalactose;6-O- galactopyranosylgalactose; 6-O-heptopyranosylglucopyranose;6-thiosucrose; 7-O-(2- amino-2-deoxyglucopyranosyl)heptose;8-methoxycarbonyloctyl-3-O-glucopyranosyl- mannopyranoside;8-O-(4-amino-4-deoxyarabinopyranosyl)-3-deoxyoctulosonic acid;allolactose; allosucrose; allyl 6-O-(3-deoxyoct-2-ulopyranosylonicacid)-(1-6)-2-deoxy-2- (3-hydroxytetradecanamido)glucopyranoside4-phosphate; alpha-(2-9)-disialic acid; alpha,alpha-trehalose6,6′-diphosphate; alpha-glucopyranosyl alpha-xylopyranoside; alpha-maltosyl fluoride; aprosulate; benzyl2-acetamido-2-deoxy-3-O-(2-O-methyl-beta-galactosyl)-beta-glucopyranoside; benzyl 2-acetamido-2-deoxy-3-O-betafucopyranosyl- alpha-galactopyranoside; benzyl2-acetamido-6-O-(2-acetamido-2,4-dideoxy-4-fluoroglucopyranosyl)-2-deoxygalactopyranoside; benzyl gentiobioside;beta-D- galactosyl(1-3)-4-nitrophenyl-N-acetyl-alpha-D-galactosamine;beta-methylmelibiose; calcium sucrose phosphate; camiglibose; cellobial;cellobionic acid; cellobionolactone; Cellobiose; cellobiose octaacetate;cellobiosyl bromide heptaacetate; Celsior; chitobiose; chondrosine;Cristolax; deuterated methyl beta-mannobioside; dextrin maltose; D-glucopyranose, O-D-glucopyranosyl; Dietary Sucrose; difructose anhydrideI; difructose anhydride III; difructose anhydride IV; digalacturonicacid; DT 5461; ediol; epilactose; epsilon-N-1-(1-deoxylactulosyl)lysine;feruloyl arabinobiose; floridoside; fructofuranosyl-(2-6)-glucopyranoside; FZ 560; galactosyl-(1-3)galactose; garamine;gentiobiose; geranyl6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside; geranyl6-O-xylopyranosyl- glucopyranoside; glucosaminyl-1,6-inositol-1,2-cyclicmonophosphate; glucosyl (1-4) N- acetylglucosamine;glucuronosyl(1-4)-rhamnose; heptosyl-2-keto-3-deoxyoctonate; inulobiose;Isomaltose; isomaltulose; isoprimeverose; kojibiose; lactobionic acid;lacto-N- biose II; Lactose; lactosylurea; Lactulose; laminaribiose;lepidimoide; leucrose; levanbiose; lucidin 3-O-beta-primveroside; LW10121; LW 10125; LW 10244; maltal; maltitol; Maltose; maltosehexastearate; maltose-maleimide; maltosylnitromethane heptaacetate;maltosyltriethoxycholesterol; maltotetraose; Malun 25; mannosucrose;mannosyl-(1-4)-N- acetylglucosaminyl-(1-N)-urea;mannosyl(2)-N-acetyl(2)-glucose; melibionic acid; Melibiose;melibiouronic acid; methyl 2-acetamido-2-deoxy-3-O-(alpha-idopyranosyluronic acid)-4-O-sulfo-beta-galactopyranoside; methyl2-acetamido-2-deoxy- 3-O-(beta-glucopyranosyluronicacid)-4-O-sulfo-beta-galactopyranoside; methyl 2-acetamido-2-deoxy-3-O-glucopyranosyluronoylglucopyranoside; methyl2-O-alpha- rhamnopyranosyl-beta-galactopyranoside; methyl2-O-beta-rhamnopyranosyl-beta- galactopyranoside; methyl2-O-fucopyranosylfucopyranoside 3 sulfate; methyl 2-O-mannopyranosylmannopyranoside; methyl2-O-mannopyranosyl-rhamnopyranoside; methyl3-O-(2-acetamido-2-deoxy-6-thioglucopyranosyl)galactopyranoside; methyl3-O- galactopyranosylmannopyranoside; methyl3-O-mannopyranosylmannopyranoside; methyl3-O-mannopyranosyltalopyranoside; methyl3-O-talopyranosyltalopyranoside; methyl 4-O-(6-deoxy-manno-heptopyranosyl)galactopyranoside; methyl6-O-acetyl-3-O-benzoyl-4-O-(2,3,4,6-tetra-O-benzoylgalactopyranosyl)-2-deoxy-2-phthalimidoglucopyranoside;methyl 6-O-mannopyranosylmannopyranoside; methyl beta-xylobioside;methyl fucopyranosyl(1- 4)-2-acetamido-2-deoxyglucopyranoside; methyllaminarabioside; methyl O-(3-deoxy-3-fluorogalactopyranosyl)(1-6)galactopyranoside; methyl-2-acetamido-2-deoxyglucopyranosyl-1-4-glucopyranosiduronic acid; methyl-2-O-fucopyranosylfucopyranoside 4-sulfate; MK 458;N(1)-2-carboxy-4,6-dinitrophenyl-N(6)- lactobionoyl-1,6-hexanediamine;N-(2,4-dinitro-5-fluorophenyl)-1,2-bis(mannos-4′- yloxy)propyl-2-amine;N-(2′-mercaptoethyl)lactamine; N-(2-nitro-4-azophenyl)-1,3-bis(mannos-4′-yloxy)propyl-2-amine;N-(4-azidosalicylamide)-1,2-bis(mannos-4′- yloxy)propyl-2-amine;N,N-diacetylchitobiose; N-acetylchondrosine; N-acetyldermosine;N-acetylgalactosaminyl-(1-4)-galactose;N-acetylgalactosaminyl-(1-4)-glucose; N-acetylgalactosaminyl-1-4-N-acetylglucosamine;N-acetylgalactosaminyl-1-4-N- acetylglucosamine;N-acetylgalactosaminyl-alpha(1-3)galactose; N-acetylglucosamine-N-acetylmuramyl-alanyl-isoglutaminyl-alanyl-glycerol dipalmitoyl;N-acetylglucosaminyl beta(1-6)N-acetylgalactosamine;N-acetylglucosaminyl-1-2-mannopyranose; N- acetylhyalobiuronic acid;N-acetylneuraminoyllactose; N-acetylneuraminoyllactose sulfate ester;N-acetylneuraminyl-(2-3)-galactose; N-acetylneuraminyl-(2-6)-galactose;N-benzyl- 4-O-(beta-galactopyranosyl)glucamine-N-carbodithioate;neoagarobiose; N- formylkansosaminyl-(1-3)-2-O-methylrhamnopyranose;O-((Nalpha)-acetylglucosamine 6- sulfate)-(1-3)-idonic acid;O-(4-O-feruloyl-alpha-xylopyranosyl)-(1-6)-glucopyranose; O-(alpha-idopyranosyluronic acid)-(1-3)-2,5-anhydroalditol-4-sulfate;O-(glucuronic acid 2- sulfate)-(1--3)-O-(2,5)-andydrotalitol 6-sulfate;O-(glucuronic acid 2-sulfate)-(1--4)-O- (2,5)-anhydromannitol 6-sulfate;O-alpha-glucopyranosyluronate-(1-2)-galactose; O-beta-galactopyranosyl-(1-4)-O-beta-xylopyranosyl-(1-0)-serine; octylmaltopyranoside; O- demethylpaulomycin A; O-demethylpaulomycin B;O-methyl-di-N-acetyl beta- chitobioside; Palatinit; paldimycin;paulomenol A; paulomenol B; paulomycin A; paulomycin A2; paulomycin B;paulomycin C; paulomycin D; paulomycin E; paulomycin F; phenyl2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-alpha-D-galactopyranoside;phenylO-(2,3,4,6-tetra-O-acetylgalactopyranosyl)-(1-3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-1-thioglucopyranoside; poly-N-4-vinylbenzyllactonamide;pseudo-cellobiose; pseudo-maltose;rhamnopyranosyl-(1-2)-rhamnopyranoside-(1-methyl ether); rhoifolin;ruberythric acid; S-3105; senfolomycin A; senfolomycin B; solabiose; SS554; streptobiosamine; Sucralfate; Sucrose; sucrose acetate isobutyrate;sucrose caproate; sucrose distearate; sucrose monolaurate; sucrosemonopalmitate; sucrose monostearate; sucrose myristate; sucroseoctaacetate; sucrose octabenzoic acid; sucrose octaisobutyrate; sucroseoctasulfate; sucrose polyester; sucrose sulfate; swertiamacroside;T-1266; tangshenoside I;tetrahydro-2-((tetrahydro-2-furanyl)oxy)-2H-pyran; thionigerose;Trehalose; trehalose 2-sulfate; trehalose 6,6′-dipalmitate;trehalose-6-phosphate; rehalulose; trehazolin; trichlorosucrose;tunicamine; turanose; U 77802; U 77803; xylobiose; xylose- glucose; andxylosucrose

Microalgae contain photosynthetic machinery capable of metabolizingphotons, and transferring energy harvested from photons into fixedchemical energy sources such as monosaccharide. Glucose is a commonmonosaccharide produced by microalgae by metabolizing light energy andfixing carbon from carbon dioxide. Some microalgae can also grow in theabsence of light on a fixed carbon source that is exogenously provided(for example see Plant Physiol. 2005 February; 137(2):460-74). Inaddition to being a source of chemical energy, monosaccharides such asglucose are also substrate for production of polysaccharides (seeExample 14). The invention provides methods of producing polysaccharideswith novel monosaccharide compositions. For example, microalgae iscultured in the presence of culture media that contains exogenouslyprovided monosaccharide, such as glucose. The monosaccharide is taken upby the cell by either active or passive transport and incorporated intopolysaccharide molecules produced by the cell. This novel method ofpolysaccharide composition manipulation can be performed with, forexample, any microalgae listed in Table 1A or Table 1B using anymonosaccharide or disaccharide listed in Tables 2 or 3.

In some embodiments, the fixed carbon source is one or more selectedfrom glucose, galactose, xylose, mannose, rhamnose, N-acetylglucosamine,glycerol, floridoside, and glucuronic acid. The methods may be practicedcell growth in the presence of at least about 5.0 μM, at least about 10μM, at least about 15.0 μM, at least about 20.0 μM, at least about 25.0μM, at least about 30.0 μM, at least about 35.0 μM, at least about 40.0μM, at least about 45.0 μM, at least about 50.0 μM, at least about 55.0μM, at least about 60.0 μM, at least about 75.0 μM, at least about 80.0μM, at least about 85.0 μM, at least about 90.0 μM, at least about 95.0μM, at least about 100.0 μM, or at least about 150.0 μM, of one or moreexogenously provided fixed carbon sources selected from Tables 2 and 3.

In some embodiments using cells of the genus Porphyridium, the methodsinclude the use of approximately 0.5-0.75% glycerol as a fixed carbonsource when the cells are cultured in the presence of light.Alternatively, a range of glycerol, from approximately 4.0% toapproximately 9.0% may be used when the Porphyridium cells are culturedin the dark, more preferably from 5.0% to 8.0%, and more preferably7.0%.

After culturing the microalgae in the presence of the exogenouslyprovided carbon source, the monosaccharide composition of thepolysaccharide can be analyzed as described in Example 5.

Microalgae culture media can contain a fixed nitrogen source such asKNO₃. Alternatively, microalgae are placed in culture conditions that donot include a fixed nitrogen source. For example, Porphyridium sp. cellsare cultured for a first period of time in the presence of a fixednitrogen source, and then the cells are cultured in the absence of afixed nitrogen source (see for example Adda M., Biomass 10: 131-140.(1986); Sudo H., et al., Current Microbiology Vol. 30 (1995), pp.219-222; Marinho-Soriano E., Bioresour Technol. 2005 February;96(3):379-82; Bioresour. Technol. 42: 141-147 (1992)). While theinvention is not limited by theory, it is well accepted by those skilledin the art that the red color of Porphyridium is due to the redpigmented light harvesting protein phycoerythrin (for example seeFujimori and Pecci, Distinct subunits of phycoerythrin from Porphyridiumcruentum and their spectral characteristics, Arch. Biochem. Biophys.118, 448-55 1967). Culture of Porphyridium in the presence of reducedlevels of nitrogen causes cells to degrade phycoerythrin, resulting in asignificant decrease in the amount of red pigmentation. Becausephycoerythrin constitutes over 2% of the dry weight of Porphyridiumcells under nitrogen-replete conditions (see for example M. M. RebollosoFuentes 2000, Food Chemistry, 70; 345-353), this catabolic processallows a significant amount of fixed nitrogen to be recycled. Againwhile the invention is not limited by theory, providing excess lightalso causes Porphyridium cells to degrade phycoerythrin to reduce theamount of light harvesting per cell. This process reduces oxidativestress caused by excess photon flux in the thylakoid membrane.Porphyridium biomass grown in nitrogen-limited conditions, particularlywhen grown under high light, lose red coloration and turn yellow withoccasional shades of light brown, referred to as “decolorized biomass”.The invention provides novel methods of production of compositions fortopical application including culturing cells of the genus Porphyridiumunder reduced levels of nitrogen (such as, for example, no Tris and lessthan 20% of the KNO₃ per liter of ATCC 1495 ASW media) and optionallyalso under relatively light conditions such as, for example, 130 μm⁻²s⁻¹. In other embodiments, the culture media contains no more than 300mg/L of one or more nitrate-containing compounds that can be metabolizedby the cells (such as, for example, but not limited to KNO₃) atinoculation, no more than 250 mg/L at inoculation, no more than 200 mg/Lat inoculation, no more than 175 mg/L at inoculation, no more than 150mg/L at inoculation, no more than 135 mg/L at inoculation, no more than125 mg/L at inoculation, no more than 50 mg/L at inoculation, no morethan 25 mg/L at inoculation, and no more than 12.5 mg/L at inoculation.In some methods the nitrate-containing compounds are provided in theculture media at inoculation at approximately 180 mg/L, approximately160 mg/L, approximately 140 mg/L, approximately 130 mg/L, approximately125 mg/L, approximately 110 mg/L, approximately 100 mg/L, andapproximately 90 mg/L. In other embodiments, the culture media containsno more than 300 millimolar of one or more nitrate-containing compoundsthat can be metabolized by the cells (such as, for example, but notlimited to KNO₃) at inoculation, no more than 250 millimolar atinoculation, no more than 200 millimolar at inoculation, no more than175 millimolar at inoculation, no more than millimolar at inoculation,no more than millimolar at inoculation, no more than millimolar atinoculation, no more than 50 millimolar at inoculation, no more than 25millimolar at inoculation, and no more than 12.5 millimolar atinoculation. In some methods the nitrate-containing compounds areprovided in the culture media at inoculation at approximately 180millimolar, approximately 160 millimolar, approximately 140 millimolar,approximately 130 millimolar, approximately 125 millimolar,approximately 110 millimolar, approximately 100 millimolar, andapproximately 90 millimolar. Inoculation can mean when seed cells areinfused into a bioreactor, and can also mean when cells grown innitrogen-replete media that have been pelleted, optionally washed, andare resuspended in culture media that has limiting amounts ofnitrogen-containing compounds or no nitrogen-containing compounds. Cellsinoculated into media containing limiting amounts of nitrogen-containingcompounds, such as 125 mg/L, will typically undergo cell division untilnitrogen is used up (assuming other nutrients are not limiting), andthen begin the “bleaching” process in which phycoerythrin is degraded.This process is accelerated as light intensity is increased. Cells grownin nitrogen-replete media can also be harvested and washed andresuspended in culture media that contains no nitrogen or a limitedamount of nitrogen such as the amounts listed above. In addition,nitrogen-replete media can be exchanged with nitrogen-limited mediathrough tangential flow filtration using a filter that has a pore sizesmaller than the diameter of the cells. The diameter of Porphyridiumcells is approximately 4-8 microns. This method avoids centrifugationand reduces the possibility of contamination.

Other methods include removing red coloration from different species ofmicroalgae through mutagenesis. For example, species of the genusPorphyridium are subjected to chemical mutagenesis, followed byscreening for colonies lacking red coloration. See for example Sivan andArad, Phycologia 32(1), pp. 68-72 (1993). Examples of novel strains ofPorphyridium sp. lacking red pigment, generated through chemicalmutagenesis as described in Sivan and Arad, are shown in FIG. 10( c).Such genetically decolorized strains are used to generate non-redbiomass for formulation as skin care products. In a preferred embodimentthe genetically decolorized biomass is homogenized. In another preferredembodiment the polysaccharide contains more than 4.75% sulfur by weight.While the invention is not limited by theory, production ofphycoerythrin is reduced in some mutagenized strains due to mutations invarious regions of the genome including promoters, coding regions, andother functional elements. Both bleaching through nutrient limitationand excess light, as well as through mutagenesis, can be performed onany microalgae species, including those listed in Tables 1A and 1B.

Some methods further comprise formulating decolorized Porphyridiumbiomass, generated through any or all of the methods nutrientlimitation, excess light, and mutagenesis, with a carrier suitable fortopical administration. The methods also optionally include formulatingdecolorized Porphyridium biomass with one or more preservatives, such asfor example diiodomethyl-p-tolylsulfone,2-Bromo-2-nitropropane-1,3-diol, cis isomer1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride (a.k.a.Dowicil 200), glutaraldehyde, 4,4-dimethyl oxazolidine,7-Ethylbicyclooxazolidine, methyl paraben, sorbic acid, methyl paraben,Germaben II, and disodium EDTA.

Other culture parameters can also be manipulated, such as the pH of theculture media, the identity and concentration of trace elements such asthose listed in Table 3, and other media constituents. One non-limitingexample is the inclusion of at least one source of sulfate in theculture media to increase the level of sulfation in the polysaccharidesproduced. In some embodiments, a polysaccharide preparation method ispracticed with culture media containing more than about 100, more thanabout 150, more than about 200, more than about 250, more than about300, more than about 350, more than about 400, more than about 450, morethan about 500, more than about 550, more than about 600, more thanabout 650, more than about 700, or more than about 750, more than about800, more than about 850, more than about 900, more than about 950, ormore than about 1 or 2 M sulfate (or total SO₄ 2). Increasing thesulfation has been demonstrated to increase the antioxidant apacity ofthe polysaccharide (see Example 23 and Spitz et al. (J. Appl. Phycology(2005) 17: 215-222). Without being bound by theory, and offered toimproved the understanding of certain aspects of the disclosedinvention, it is possible that an increased level of sulfation mayincrease the anti-cholesterol characteristics of the homogenized cellmaterial or polysaccharide preparation disclosed herein. The correlationbetween higher amounts of sulfation and antioxidant activitydemonstrated herein was unexpected given the weak antioxidant activityof carrageenan, which contains as much as 40% sulfate.

It is believed that microalgae of the genus Porphyridium have not beengrown or propagated under conditions with sulfate concentrations of 100mM to 2 M. Thus the invention includes the surprising discovery thatmicroalgae are capable of growth under such conditions. Additionally,the invention is based in part on the surprising discovery that growthunder higher sulfate concentrations can produce polysaccharides withhigher levels of sulfation. This allows for the production of cells (andso biomass) containing highly sulfated polysaccharides that may be usedin the form of purified polysaccharides, a homogenate of cells(biomass), intact cells (biomass) per se, or a combination thereof.

The discovery that Porphyridium can survive above 100 mM sulfate wassurprising for a number of reasons. First, it is known that sulfate canalter the rate of uptake of toxic metal ions in algae, such as chromium,and that increasing metal accumulation can lead to toxicity (see forexample Kaszycki et al, Plant, Cell & Environment, 28(2): p. 260, 2005).Second, it is also known that sulfate can inhibit the uptake of metalions required for nitrogen fixation such as molybdenum, and thatincreasing sulfate concentrations negatively affects algae such ascyanobacteria even at sulfate concentrations in estuarine (>8-10 mM) andseawater (28 mM) levels of sufate (see for example Marino et al,Hydrobiologia 500: pp. 277-293, 2004). Third, sulfate at high levels canoften be taken up and reduced and sulfide, which is toxic tophotosynthesis because it attacks photosystem II (see for example Khanalet al., J. Envir. Engrg., 129(12); pp. 1104-1111, 2003). Fourth, highsulfate levels alter the osmotic pressure of the growth media, and manyorganisms cannot survive at such an elevated osmoticum. For example, itis well established that photosynthesis of algae is inhibited byhyperosmotic and salt stresses. See for example “Suppression of QuantumYield of Photosystem II by Hyperosmotic Stress in Chlamydomonasreinhardtii” Plant Cell Physiol. 36: pp. 1253-1258 (1995); and “TheEffect of Osmotic and Ionic Stress on the Primary Processes ofPhotosynthesis in Dunaliella tertiolecta”, J. Exp. Bot. 1984 35(1):18-27 (1984).

By use of methods described above, the disclosed invention includes apreparation of cells, cell biomass, or cell homogenate containing areduced level of green pigmentation, or a reduced absorbance at 545 nm,relative to the same cells grown under different conditions. The cellsmay be those of any microalgae as described herein, including those ofthe genus Porphyridium. The cells, cell biomass, or cell homogenate maybe formulated into a composition of the disclosure such that an aqueousextract of the composition would contain the same reduced level of greenpigmentation.

In some embodiments, an aqueous extract of the composition contains nomore than about 75%, no more than about 70%, no more than about 65%, nomore than about 60%, no more than about 55%, no more than about 50%, nomore than about 45%, no more than about 40%, no more than about 35%, nomore than about 30%, no more than about 25%, no more than about 20%, nomore than about 15%, no more than about 10%, or no more than about 5% ofthe absorbance per gram at 545 nm compared to an extract of cells of thesame species grown in a photobioreactor in ATCC 1495 ASW media (asdescribed in Example 1) in the presence of 50 microeinsteins of lightper second per square meter. One non-limiting means for detection ofabsorbance is by use of a spectrophotometer.

Microalgae can be grown in the presence of light. The number of photonsstriking a culture of microalgae cells can be manipulated, as well asother parameters such as the wavelength spectrum and ratio of dark:lighthours per day. Microalgae can also be cultured in natural light, as wellas simultaneous and/or alternating combinations of natural light andartificial light. For example, microalgae of the genus Chlorella becultured under natural light during daylight hours and under artificiallight during night hours.

The gas content of a photobioreactor can be manipulated. Part of thevolume of a photobioreactor can contain gas rather than liquid. Gasinlets can be used to pump gases into the photobioreactor. Any gas canbe pumped into a photobioreactor, including air, air/CO₂ mixtures, noblegases such as argon and others. The rate of entry of gas into aphotobioreactor can also be manipulated. Increasing gas flow into aphotobioreactor increases the turbidity of a culture of microalgae.Placement of ports conveying gases into a photobioreactor can alsoaffect the turbidity of a culture at a given gas flow rate. Air/CO₂mixtures can be modulated to generate different polysaccharidecompositions by manipulating carbon flux. For example, air:CO₂ mixturesof about 99.75% air:0.25% CO₂, about 99.5% air:0.5% CO₂, about 99.0%air:1.00% CO₂, about 98.0% air:2.0% CO₂, about 97.0% air:3.0% CO₂, about96.0% air:4.0% CO₂, and about 95.00% air:5.0% CO₂ can be infused into abioreactor or photobioreactor.

Microalgae cultures can also be subjected to mixing using devices suchas spinning blades and propellers, rocking of a culture, stir bars, andother instruments.

Microalgae can be cultured in liquid media. The culture can be containedwithin a bioreactor. Optionally, the bioreactor does not allow light toenter. Alternatively, microalgae can also be cultured inphotobioreactors that contain a fixed carbon source and allow light tostrike the cells. Exposure of microalgae cells to light, even in thepresence of a fixed carbon source that the cells transport and utilize(i.e., mixotrophic growth), nonetheless accelerates growth compared toculturing cells in the dark. Culture condition parameters can bemanipulated to optimize total hydrocarbon production, the combination ofhydrocarbon species produced, and/or production of a hydrocarbonspecies. In some instances it is preferable to culture cells in thedark, such as, for example, when using extremely large (40,000 liter andhigher) fermentors that do not allow light to strike the culture.

Microalgal culture media typically contains components such as a fixednitrogen source, trace elements, optionally a buffer for pH maintenance,and phosphate. Other components can include a fixed carbon source suchas acetate or glucose, and salts such as sodium chloride, particularlyfor seawater microalgae. Examples of trace elements include zinc, boron,cobalt, copper, manganese, and molybdenum in, for example, therespective forms of ZnCl₂, H₃BO₃, CoCl₂.6H₂O, CuCl₂.2H₂O, MnCl₂.4H₂O and(NH₄)₆Mo₇O₂.4H₂O.

For organisms able to grow on a fixed carbon source, the fixed carbonsource can be, for example, glucose, fructose, sucrose, galactose,xylose, mannose, rhamnose, N-acetylglucosamine, glycerol, floridoside,and/or glucuronic acid. The one or more carbon source(s) can be suppliedat a concentration of at least about 50 μM, at least about 100 μM, atleast about 500 μM, at least about 5 mM, at least about 50 mM, and atleast about 500 mM, of one or more exogenously provided fixed carbonsource(s). Some microalgae species can grow by utilizing a fixed carbonsource such as glucose or acetate in the absence of light. Such growthis known as heterotrophic growth.

Other culture parameters can also be manipulated, such as the pH of theculture media, the identity and concentration of trace elements andother media constituents.

B. Cell Culture Methods: Photobioreactors

Microalgae can be grown and maintained in closed photobioreactors madeof different types of transparent or semitransparent material. Suchmaterial can include Plexiglas® enclosures, glass enclosures, bags badefrom substances such as polyethylene, transparent or semitransparentpipes, and other materials. Microalgae can also be grown in open ponds.

Photobioreactors can have ports allowing entry of gases, solids,semisolids and liquids into the chamber containing the microalgae. Portsare usually attached to tubing or other means of conveying substances.Gas ports, for example, convey gases into the culture. Pumping gasesinto a photobioreactor can serve to both feed cells CO₂ and other gasesand to aerate the culture and therefore generate turbidity. The amountof turbidity of a culture varies as the number and position of gas portsis altered. For example, gas ports can be placed along the bottom of acylindrical polyethylene bag. Microalgae grow faster when CO₂ is addedto air and bubbled into a photobioreactor. For example, a 5% CO₂:95% airmixture is infused into a photobioreactor containing cells of the genusPorphyridium (see for example Biotechnol Bioeng. 1998 Sep. 20; 59(6):705-13; textbook from office; U.S. Pat. Nos. 5,643,585 and 5,534,417;Lebeau, T., et. al. Appl. Microbiol. Biotechnol (2003) 60: 612-623;Muller-Fuega, A., Journal of Biotechnology 103 (2003 153-163;Muller-Fuega, A., Biotechnology and Bioengineering, Vol. 84, No. 5, Dec.5, 2003; Garcia-Sanchez, J. L., Biotechnology and Bioengineering, Vol.84, No. 5, Dec. 5, 2003; Garcia-Gonzales, M., Journal of Biotechnology,115 (2005) 81-90. Molina Grima, E., Biotechnology Advances 20 (2003)491-515).

Photobioreactors can be exposed to one or more light sources to providemicroalgae with light as an energy source via light directed to asurface of the photobioreactor. Preferably the light source provides anintensity that is sufficient for the cells to grow, but not so intenseas to cause oxidative damage or cause a photoinhibitive response. Insome instances a light source has a wavelength range that mimics orapproximately mimics the range of the sun. In other instances adifferent wavelength range is used. Photobioreactors can be placedoutdoors or in a greenhouse or other facility that allows sunlight tostrike the surface. Photon intensities are typically measured inmicroeinsteins of light per square meter per second (uE m⁻² s⁻¹)although other measurements such as lux and footcandles are sometimesused. Preferred photon intensities for culturing species of the genusPorphyridium are between 50 and 300 uE m⁻² s⁻¹ (see for examplePhotosynth Res. 2005 June; 84(1-3): 21-7), although in cases of inducingPorphyridium cells to degrade phycoerythrin preferred light intensitiescan be higher, such as for example 400-700 uE m⁻²s⁻¹.

Photobioreactors preferably have one or more parts that allow mediaentry. It is not necessary that only one substance enter or leave aport. For example, a port can be used to flow culture media into thephotobioreactor and then later can be used for sampling, gas entry, gasexit, or other purposes. In some instances a photobioreactor is filledwith culture media at the beginning of a culture and no more growthmedia is infused after the culture is inoculated. In other words, themicroalgal biomass is cultured in an aqueous medium for a period of timeduring which the microalgae reproduce and increase in number; howeverquantities of aqueous culture medium are not flowed through thephotobioreactor throughout the time period. Thus in some embodiments,aqueous culture medium is not flowed through the photobioreactor afterinoculation.

In other instances culture media can be flowed though thephotobioreactor throughout the time period during which the microalgaereproduce and increase in number. In some instances media is infusedinto the photobioreactor after inoculation but before the cells reach adesired density. In other words, a turbulent flow regime of gas entryand media entry is not maintained for reproduction of microalgae until adesired increase in number of said microalgae has been achieved, butinstead a parameter such as gas entry or media entry is altered beforethe cells reach a desired density.

Photobioreactors preferably have one or more ports that allow gas entry.Gas can serve to both provide nutrients such as CO₂ as well as toprovide turbulence in the culture media. Turbulence can be achieved byplacing a gas entry port below the level of the aqueous culture media sothat gas entering the photobioreactor bubbles to the surface of theculture. One or more gas exit ports allow gas to escape, therebypreventing pressure buildup in the photobioreactor. Preferably a gasexit port leads to a “one-way” valve that prevents contaminatingmicroorganisms to enter the photobioreactor. In some instances cells arecultured in a photobioreactor for a period of time during which themicroalgae reproduce and increase in number, however a turbulent flowregime with turbulent eddies predominantly throughout the culture mediacaused by gas entry is not maintained for all of the period of time. Inother instances a turbulent flow regime with turbulent eddiespredominantly throughout the culture media caused by gas entry can bemaintained for all of the period of time during which the microalgaereproduce and increase in number. In some instances a predeterminedrange of ratios between the scale of the photobioreactor and the scaleof eddies is not maintained for the period of time during which themicroalgae reproduce and increase in number. In other instances such arange can be maintained.

Photobioreactors preferably have at least one port that can be used forsampling the culture. Preferably a sampling port can be used repeatedlywithout altering compromising the axenic nature of the culture. Asampling port can be configured with a valve or other device that allowsthe flow of sample to be stopped and started. Alternatively a samplingport can allow continuous sampling. Photobioreactors preferably have atleast one port that allows inoculation of a culture. Such a port canalso be used for other purposes such as media or gas entry.

Microalgae that produce polysaccharides can be cultured inphotobioreactors. Microalgae that produce polysaccharide that is notattached to cells can be cultured for a period of time and thenseparated from the culture media and secreted polysaccharide by methodssuch as centrifugation and tangential flow filtration. Preferredorganisms for culturing in photobioreactors to produce polysaccharidesinclude Porphyridium sp., Porphyridium cruentum, Porphyridium purpureum,Porphyridium aerugineum, Rhodella maculata, Rhodella reticulata,Chlorella autotrophica, Chlorella stigmatophora, Chlorella capsulata,Achnanthes brevipes, Achnanthes longipes, Gloeocapsa alpicola andPhaeocysstis pouchettii.

C. Cell Culture Methods: Heterotrophic Growth

As an alternative to photosynthetic growth of microorganisms, asdescribed above, some microorganisms can be cultured under heterotrophicgrowth conditions in which a fixed carbon source provides energy forgrowth and polysaccharide production.

For polysaccharide production, cells, including recombinant cells of theinvention described herein, are preferably cultured or fermented inlarge quantities. The culturing may be in large liquid volumes, such asin suspension cultures as an example. Other examples include startingwith a small culture of cells which expand into a large biomass incombination with cell growth and propagation as well as polysaccharideproduction. Bioreactors or steel fermentors can be used to accommodatelarge culture volumes. A fermentor similar those used in the productionof beer and/or wine is suitable, as are extremely large fermentors usedin the production of ethanol.

Appropriate nutrient sources for culture in a fermentor are provided.These include raw materials such as one or more of the following: afixed carbon source such as glucose, corn starch, depolymerizedcellulosic material, sucrose, sugar cane, sugar beet, lactose, milkwhey, or molasses; a fat source, such as fats or vegetable oils; anitrogen source, such as protein, soybean meal, cornsteep liquor,ammonia (pure or in salt form), nitrate or nitrate salt, or molecularnitrogen; and a phosphorus source, such as phosphate salts.Additionally, a fermentor allows for the control of culture conditionssuch as temperature, pH, oxygen tension, and carbon dioxide levels.Optionally, gaseous components, like oxygen or nitrogen, can be bubbledthrough a liquid culture. Other Starch (glucose) sources such as wheat,potato, rice, and sorghum. Other carbon sources include process streamssuch as technical grade glycerol, black liquor, organic acids such asacetate, and molasses. Carbon sources can also be provided as a mixture,such as a mixture of sucrose and depolymerized sugar beet pulp.

A fermentor can be used to allow cells to undergo the various phases oftheir growth cycle. As an example, an inoculum ofpolysaccharide-producing cells can be introduced into a medium followedby a lag period (lag phase) before the cells begin growth. Following thelag period, the growth rate increases steadily and enters the log, orexponential, phase. The exponential phase is in turn followed by aslowing of growth due to decreases in nutrients and/or increases intoxic substances. After this slowing, growth stops, and the cells entera stationary phase or steady state, depending on the particularenvironment provided to the cells.

In an alternate heterotrophic growth method in accordance with thepresent invention, microorganisms can be cultured using depolymerizedcellulosic biomass as a feedstock. Cellulosic biomass (e.g., stover,such as corn stover) is inexpensive and readily available; however,attempts to use this material as a feedstock for yeast have failed. Inparticular, such feedstock have been found to be inhibitory to yeastgrowth, and yeast cannot use the 5-carbon sugars produced fromcellulosic materials (e.g., xylose from hemi-cellulose). By contrast,microalgae can grow on processed cellulosic material. Accordingly, theinvention provides a method of culturing a microalgae in the presence ofa cellulosic material and/or a 5-carbon sugar. Cellulosic materialsgenerally include:

Component Percent Dry Weight Cellulose 40-60% Hemicellulose 20-40%Lignin 10-30%

Suitable cellulosic materials include residues from herbaceous and woodyenergy crops, as well as agricultural crops, i.e., the plant parts,primarily stalks and leaves, not removed from the fields with theprimary food or fiber product. Examples include agricultural wastes suchas sugarcane bagasse, rice hulls, corn fiber (including stalks, leaves,husks, and cobs), wheat straw, rice straw, sugar beet pulp, citrus pulp,citrus peels; forestry wastes such as hardwood and softwood thinnings,and hardwood and softwood residues from timber operations; wood wastessuch as saw mill wastes (wood chips, sawdust) and pulp mill waste; urbanwastes such as paper fractions of municipal solid waste, urban woodwaste and urban green waste such as municipal grass clippings; and woodconstruction waste. Additional cellulosics include dedicated cellulosiccrops such as switchgrass, hybrid poplar wood, and miscanthus, fibercane, and fiber sorghum. Five-carbon sugars that are produced from suchmaterials include xylose.

In still another alternative heterotrophic growth method in accordancewith the present invention, which itself may optionally be used incombination with the methods described above, sucrose, produced byexample from sugar cane or sugar beet, is used as a feedstock.Microalgae can be engineered to utilize sucrose as a carbon source. Forexample, expression of a sucrose transporter and a sucrose invertaseallows microalgae, e.g., Chlorella protothecoides, to transport sucroseinto the cell from the culture media and hydrolyze sucrose to yieldglucose and fructose. Optionally, a fructokinase can be expressed aswell in instances where endogenous hexokinase activity is insufficientfor maximum phosphorylation of fructose. Examples of suitable sucrosetransporters are Genbank accession numbers CAD91334, CAB92307, andCAA53390. Examples of suitable sucrose invertases are Genbank accessionnumbers CAB95010, NP_(—)012104 and CAA06839. Examples of suitablefructokinases are Genbank accession numbers P26984, P26420 and CAA43322.Vectors for transformation of microalgae, including Chlorella, encodingone or more of such genes can be designed as described herein, or asdescribed in U.S. patent application Ser. No. 12/131,783, filed Jun. 2,2008, titled “Use of Cellulosic Materials for Cultivation ofMicroorganisms,” and incorporated herein by reference in its entirety.

Secretion of a sucrose invertase can obviate the need for expression ofa transporter that can transport sucrose into the cell. This is becausea secreted invertase catalyzes the conversion of a molecule of sucroseinto a molecule of glucose and a molecule of fructose, both of which canbe transported and utilized by microbes disclosed herein. For example,expression of a sucrose invertase with a secretion signal generatesinvertase activity outside the cell. See Hawkins et al., CurrentMicrobiology Vol. 38 (1999), pp. 335-341 for examples of secretionsignals active in Chlorella. Expression of such a protein, as enabled bythe genetic engineering methodology disclosed herein, allows cellsalready capable of utilizing extracellular glucose as an energy sourceto utilize sucrose as an extracellular energy source.

Alternatively, a sucrose invertase can also be expressed intracellularlyin cells that express a sucrose transporter, as well as in cells thatexpress any carbohydrate transporter that allows sucrose to enter thecell.

Bioreactors can be employed for use in heterotrophic growth methods. Aswill be appreciated, provisions made to make light available to thecells in photosynthetic growth methods are unnecessary when using afixed-carbon source in the heterotrophic growth methods describedherein.

The specific examples of process conditions and heterotrophic growthmethods described herein can be combined in any suitable manner toimprove efficiencies of microbial growth and lipid production. Inaddition, the invention includes the selection and/or geneticengineering of microbes, such as microalgae, to produce microbes thatare even more suitable for use in the above-described methods. Forexample, the microbes having a greater ability to utilize any of theabove-described feedstocks for increased proliferation and/orpolysaccharide production are within the scope of the invention.

D. Mixotrophic Growth

Mixotrophic growth is the use of both light and fixed carbon source(s)as energy sources for cells to grow and produce polysaccharides.Mixotrophic growth can be conducted in a photobioreactor. Microalgae canbe grown and maintained in closed photobioreactors made of differenttypes of transparent or semitransparent material. Such material caninclude Plexiglas® enclosures, glass enclosures, bags made fromsubstances such as polyethylene, transparent or semitransparent pipes,and other materials. Microalgae can be grown and maintained in openphotobioreactors such as raceway ponds, settling ponds, and othernon-enclosed containers.

E. Growth Media

Microorganisms useful in accordance with the methods of the presentinvention are found in various locations and environments throughout theworld. As a consequence of their isolation from other species and theirresulting evolutionary divergence, the particular growth medium foroptimal growth and generation of lipid and/or hydrocarbon constituentscan be difficult to predict. In some cases, certain strains ofmicroorganisms may be unable to grow on a particular growth mediumbecause of the presence of some inhibitory component or the absence ofsome essential nutritional requirement required by the particular strainof microorganism.

Solid and liquid growth media are generally available from a widevariety of sources, and instructions for the preparation of particularmedia that is suitable for a wide variety of strains of microorganismscan be found, for example, online at http://www.utex.org/, a sitemaintained by the University of Texas at Austin for its culturecollection of algae (UTEX).

In a particular example, a medium suitable for culturing Chlorellaprotothecoides (UTEX 31) comprises Proteose Medium. This medium issuitable for axenic cultures, and a 1 L volume of the medium (pH 6.8)can be prepared by addition of 1 g of proteose peptone to 1 liter ofBristol Medium. Bristol medium comprises 2.94 mM NaNO₃, 0.17 mMCaCl₂.2H₂O, 0.3 mM MgSO₄.7H₂O, 0.43 mM, 1.29 mM KH₂PO₄, and 1.43 mM NaClin an aqueous solution. For 1.5% agar medium, 15 g of agar can be addedto 1 L of the solution. The solution is covered and autoclaved, and thenstored at a refrigerated temperature prior to use.

Other suitable media for use with the methods of the invention can bereadily identified by consulting the URL identified above, or byconsulting other organizations that maintain cultures of microorganisms,such as SAG, CCAP, or CCALA. SAG refers to the Culture Collection ofAlgae at the University of Göttingen (Göttingen, Germany), CCAP refersto the culture collection of algae and protozoa managed by the ScottishAssociation for Marine Science (Scotland, United Kingdom), and CCALArefers to the culture collection of algal laboratory at the Institute ofBotany (Trebon, Czech Republic).

F. Non-Microalgal Polysaccharide Production

Organisms besides microalgae can be used to produce polysaccharides,such as lactic acid bacteria (see for example Stinglee, F., MolecularMicrobiology (1999) 32(6), 1287-1295; Ruas_Madiedo, P., J. Dairy Sci.88: 843-856 (2005); Laws, A., Biotechnology Advances 19 (2001) 597-625;Xanthan gum bacteria: Pollock, T J., J. Ind. Microbiol. Biotechnol.,1997 August; 19(2):92-7; Becker, A., Appl. Micrbiol. Bio/technol. 1998August; 50(2):92-7; Garcia-Ochoa, F., Biotechnology Advances 18 (2000)549-579, seaweed: Talarico, L B., et al., Antiviral Research 66 (2005)103-110; Dussealt, J., et al., J Biomed Mater Res A., (2005) Novl; Melo,F. R., J Biol Chem 279: 20824-35 (2004)).

G. Ex Vivo Methods

Microalgae and other organisms can be manipulated to producepolysaccharide molecules that are not naturally produced by methods suchas feeding cells with monosaccharides that are not produced by the cells(Nature. 2004 Aug. 19; 430(7002): 873-7). For example, species listed inTable 1A are grown according to the referenced growth protocols, withthe additional step of adding to the culture media a fixed carbon sourcethat is not in the culture media as published and referenced in Table 1Aand is not produced by the cells in a detectable amount.

H. In Vitro Methods

Polysaccharides can be altered by enzymatic and chemical modification.For example, carbohydrate modifying enzymes can be added to apreparation of polysaccharide and allowed to catalyze reactions thatalter the structure of the polysaccharide. Chemical methods can be usedto, for example, modify the sulfation pattern of a polysaccharide (seefor example Carbohydr. Polym. 63: 75-80 (2000); Pomin V H.,Glycobiology. 2005 December; 15(12):1376-85; Naggi A., Semin ThrombHemost. 2001 October; 27(5):437-43 Review; Habuchi, O., Glycobiology.1996 January; 6(1); 51-7; Chen, J., J. Biol. Chem. In press; Geresh., Set al., J. Biochem. Biophys. Methods 50 (2002) 179-187).

I. Polysaccharide Purification Methods

Exopolysaccharides can be purified from microalgal cultures by variousmethods, including those disclosed herein.

1. Precipitation

For example, polysaccharides can be precipitated by adding compoundssuch as cetylpyridinium chloride, isopropanol, ethanol, or methanol toan aqueous solution containing a polysaccharide in solution. Pellets ofprecipitated polysaccharide can be washed and resuspended in water,buffers such as phosphate buffered saline or Tris, or other aqueoussolutions (see for example Farias, W. R. L., et al., J. Biol. Chem.(2000) 275; (38)29299-29307; U.S. Pat. No. 6,342,367; U.S. Pat. No.6,969,705).

2. Dialysis

Polysaccharides can also be dialyzed to remove excess salt and othersmall molecules (see for example Gloaguen, V., et al., Carbohydr Res.2004 Jan. 2; 339(1): 97-103; Microbiol Immunol. 2000; 44(5): 395-400).

3. Tangential Flow Filtration

Filtration can be used to concentrate polysaccharide and remove salts.For example, tangential flow filtration (TFF), also known as cross-flowfiltration, can be used)). For a preferred filtration method see Geresh,Carb. Polym. 50; 183-189 (2002), which discusses use of a MaxCell A/Gtechnologies 0.45 uM hollow fiber filter. Also see for example MilliporePellicon® devices, used with 100 kD, 300 kD, 1000 kD (catalog numberP2C01MC01), 0.1 uM (catalog number P2VVPPV01), 0.22 uM (catalog numberP2GVPPV01), and 0.45 uM membranes (catalog number P2HVMPV01). It ispreferred that the polysaccharides do not pass through the filter at asignificant level. It is also preferred that polysaccharides do notadhere to the filter material. TFF can also be performed using hollowfiber filtration systems.

Non-limiting examples of tangential flow filtration include use of afilter with a pore size of at least about 0.1 micrometer, at least about0.12 micrometer, at least about 0.14 micrometer, at least about 0.16micrometer, at least about 0.18 micrometer, at least about 0.2micrometer, at least about 0.22 micrometer, or at least about 0.45micrometer. Preferred pore sizes of TFF allow contaminants to passthrough but not polysaccharide molecules.

4. Ion Exchange Chromatography

Anionic polysaccharides can be purified by anion exchangechromatography. (Jacobsson, I., Biochem J. 1979 Apr. 1; 179(1): 77-89;Karamanos, N K., Eur J. Biochem. 1992 Mar. 1; 204(2): 553-60).

5. Protease Treatment

Polysaccharides can be treated with proteases to degrade contaminatingproteins. In some instances the contaminating proteins are attached,either covalently or noncovalently to polysaccharides. In otherinstances the polysaccharide molecules are in a preparation that alsocontains proteins. Proteases can be added to polysaccharide preparationscontaining proteins to degrade proteins (for example, the protease fromStreptomyces griseus can be used (SigmaAldrich catalog number P5147).After digestion, the polysaccharide is preferably purified from residualproteins, peptide fragments, and amino acids. This purification can beaccomplished, for example, by methods listed above such as dialysis,filtration, and precipitation.

Heat treatment can also be used to eliminate proteins in polysaccharidepreparations (see for example Biotechnol Lett. 2005 January; 27(1):13-8;FEMS Immunol Med. Microbiol. 2004 Oct. 1; 42(2): 155-66; Carbohydr Res.2000 Sep. 8; 328(2): 199-207; J Biomed Mater Res. 1999; 48(2): 111-6;Carbohydr Res. 1990 Oct. 15; 207(1): 101-20).

The invention thus includes production of an exopolysaccharidecomprising separating the exopolysaccharide from contaminants afterproteins attached to the exopolysaccharide have been degraded ordestroyed. The proteins may be those attached to the exopolysaccharideduring culture of a microalgal cell in media, which is first separatedfrom the cells prior to proteolysis or protease treatment. The cells maybe those of the genus Porphyridium as a non-limiting example.

In one non-limiting example, a method of producing an exopolysaccharideis provided wherein the method comprises culturing cells of the genusPorphyridium; separating cells from culture media; destroying proteinattached to the exopolysaccharide present in the culture media; andseparating the exopolysaccharide from contaminants. In some methods, thecontaminant(s) are selected from amino acids, peptides, proteases,protein fragments, and salts. In other methods, the contaminant isselected from NaCl, MgSO₄, MgCl₂, CaCl₂, KNO₃, KH₂PO₄, NaHCO₃, Tris,ZnCl₂, H₃BO₃, CoCl₂, CuCl₂, MnCl₂, (NH₄)₆Mo₇O₂₄, FeCl₃ and EDTA.

6. Whole Cell Extraction

Intracellular polysaccharides and cell wall polysaccharides can bepurified from whole cell mass (see form example U.S. Pat. No. 4,992,540;U.S. Pat. No. 4,810,646; J Sietsma J H., et al., Gen Microbiol. 1981July; 125(1):209-12; Fleet G H, Manners D J., J Gen Microbiol. 1976 May;94(1):180-92).

J. Drying Methods

After purification of methods such as those above, polysaccharides canbe dried using methods such as lyophilization and heat drying (see forexample Shastry, S., Brazilian Journal of Microbiology (2005) 36: 57-62;Matthews K H., Int J. Pharm. 2005 Jan. 31; 289(1-2): 51-62. Epub 2004Dec. 30; Gloaguen, V., et al., Carbohydr Res. 2004 Jan. 2; 339(1):97-103).

Tray dryers accept moist solid on trays. Hot air (or nitrogen) can becirculated to dry. Shelf dryers can also employ reduced (belowatmospheric at sea level, such as at about 25 in Hg or less) pressure orvacuum to dry at room temperature when products are temperaturesensitive and are similar to a freeze-drier but less costly to use andcan be easily scaled-up. In some embodiments drying in oven tray dryersis performed under vacuum.

Spray dryers are relatively simple in operation, which accept feed influid state and convert it into a dried particulate form by spraying thefluid into a hot drying medium.

Rotary dryers operate by continuously feeding wet material, which isdried by contact with heated air, while being transported along theinterior of a rotating cylinder, with the rotating shell acting as theconveying device and stirrer.

Spin flash dryers are used for drying of wet cake, slurry, or pastewhich is normally difficult to dry in other dryers. The material is fedby a screw feeder through a variable speed drive into the verticaldrying chamber where it is heated by air and at the same timedisintegrated by a specially designed disintegrator. The heating of airmay be direct or indirect depending upon the application. The dry powderis collected through a cyclone separator/bag filter or with acombination of both.

K. Microalgae Homogenization Methods

A pressure disrupter pumps a slurry through a restricted orifice valve.High pressure (up to 1500 bar) is applied, followed by an instantexpansion through an exiting nozzle. Cell disruption is accomplished bythree different mechanisms: impingement on the valve, high liquid shearin the orifice, and sudden pressure drop upon discharge, causing anexplosion of the cell. The method is applied mainly for the release ofintracellular molecules. According to Hetherington et al., celldisruption (and consequently the rate of protein release) is afirst-order process, described by the relation: log[Rm/(Rm−R)]=K NP72.9. R is the amount of soluble protein; Rm is the maximum amount ofsoluble protein K is the temperature dependent rate constant; N is thenumber of passes through the homogenizer (which represents the residencetime). P is the operating pressure.

In a ball mill, cells are agitated in suspension with small abrasiveparticles. Cells break because of shear forces, grinding between beads,and collisions with beads. The beads disrupt the cells to releasebiomolecules. The kinetics of biomolecule release by this method is alsoa first-order process.

Another widely applied method is the cell lysis with high frequencysound that is produced electronically and transported through a metallictip to an appropriately concentrated cellular suspension, ie:sonication. The concept of ultrasonic disruption is based on thecreation of cavities in cell suspension. Homogenization can also beperformed with a Microfluidizer® device (such as the M-110YMicrofluidizer® model, Microfluidics Inc., Newton, Mass.).

Blending (high speed or Waring), the french press, or evencentrifugation in the case of weak cell walls, also disrupt the cells byusing the same concepts.

Cells can also be ground after drying in devices such as a colloid mill.

Because the percentage of polysaccharide as a function of the dry weightof a microalgae cell can frequently be in excess of 50%, microalgae cellhomogenates can be considered partially pure polysaccharidecompositions. Cell disruption aids in increasing the amount ofsolvent-accessible polysaccharide by breaking apart cell walls that arelargely composed of polysaccharide.

Homogenization as described herein can increase the amount ofsolvent-available polysaccharide significantly. For example,homogenization can increase the amount of solvent-availablepolysaccharide by at least a factor of 0.25, at least a factor of 0.5,at least a factor of 1, at least a factor of 2, at least a factor of 3,at least a factor of 4, at least a factor of 5, at least a factor of 8,at least a factor of 10, at least a factor of 15, at least a factor of20, at least a factor of 25, and at least a factor of 30 or morecompared to the amount of solvent-available polysaccharide in anidentical or similar quantity of non-homogenized cells of the same type.One way of determining a quantity of cells sufficient to generate agiven quantity of homogenate is to measure the amount of a compound inthe homogenate and calculate the gram quantity of cells required togenerate this amount of the compound using known data for the amount ofthe compound per gram mass of cells. The quantity of many such compoundsper gram of particular microalgae cells are know. For examples, see FIG.7. Given a certain quantity of a compound in a composition, the skilledartisan can determine the number of grams of intact cells necessary togenerate the observed amount of the compound. The number of grams ofmicroalgae cells present in the composition can then be used todetermine if the composition contains at least a certain amount ofsolvent-available polysaccharide sufficient to indicate whether or notthe composition contains homogenized cells, such as for example fivetimes the amount of solvent-available polysaccharide present in asimilar or identical quantity of unhomogenized cells.

L. Analysis Methods

Assays for detecting polysaccharides can be used to quantitate startingpolysaccharide concentration, measure yield during purification,calculate density of secreted polysaccharide in a photobioreactor,measure polysaccharide concentration in a finished product, and otherpurposes.

The phenol: sulfuric acid assay detects carbohydrates (see Hellebust,Handbook of Phycological Methods, Cambridge University Press, 1978; andCuesta G., et al., J Microbiol Methods. 2003 January; 52(1):69-73). The1,6 dimethylmethylene blue assay detects anionic polysaccharides. (seefor example Braz J Med Biol Res. 1999 May; 32(5):545-50; Clin Chem. 1986November; 32(11):2073-6).

Polysaccharides can also be analyzed through methods such as HPLC, sizeexclusion chromatography, and anion exchange chromatography (see forexample Prosky L, Asp N, Schweizer T F, DeVries J W & Furda 1 (1988)Determination of insoluble, soluble and total dietary fiber in food andfood products: Interlaboratory study. Journal of the Association ofOfficial Analytical Chemists 71, 101711023; Int J Biol Macromol. 2003November; 33(1-3): 9-18)

Polysaccharides can also be detected using gel electrophoresis (see forexample Anal Biochem. 2003 Oct. 15; 321(2): 174-82; Anal Biochem. 2002Jan. 1; 300(1): 53-68).

Monosaccharide analysis of polysaccharides can be performed by combinedgas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl(TMS) derivatives of the monosaccharide methyl glycosides produced fromthe sample by acidic methanolysis (see Merkle and Poppe (1994) MethodsEnzymol. 230: 1-15; York, et al. (1985) Methods Enzymol. 118: 3-40).

The determination of protein concentration may be by use of any knownprocedure, such as the Lowry assay, the Biuret assay, the Bradfordassay, or the bicinchoninic acid (BCA) assay. As a non-limiting example,the BCA assay is based on the formation of a Cu²⁺-protein complex underalkaline conditions. The Cu²⁺ is then reduced to Cu¹⁺ where the amountof protein present is proportional to the amount reduction. Thereduction has been shown to be mediated by amino acids such as cysteine,cystine, tryptophan, and tyrosine as well as the peptide bond. Theresult of the assay is a purple-blue complex with Cu¹⁺ under alkalineconditions. The color complex is stable, even in the presence of othercomponents possibly present with the proteins, such as detergents. Theamount of reduction can be monitored by absorbance at 562 nm. The BCAassay is sensitive and accurate over a broad range of proteinconcentrations.

IV. Compositions

Compositions of the invention include a microalgal polysaccharide orhomogenate as described herein. In embodiments relating topolysaccharides, including exopolysaccharides, the composition maycomprise a homogenous or a heterogeneous population of polysaccharidemolecules, including sulfated polysaccharides as non-limitingembodiments. Non-limiting examples of homogenous populations includethose containing a single type of polysaccharide molecule, such as thatwith the same structure and molecular weight. Non-limiting examples ofheterogeneous populations include those containing more than one type ofpolysaccharide molecule, such as a mixture of polysaccharides having amolecular weight (MW) within a range or a MW above or below a MW value.In some cases, the polysaccharide has an average molecular weight above2 million Daltons. For example, the Porphyridium sp. exopolysaccharideis typically produced in a range of sizes from 3-5 million Daltons. Ofcourse a polysaccharide containing composition of the invention may beoptionally protease treated, or reduced in the amount of protein, asdescribed above.

In some embodiments, a composition of the invention may comprise one ormore polysaccharides produced by microalgae that have not beenrecombinantly modified. The microalgae may be those which are naturallyoccurring or those which have been maintained in culture in the absenceof alteration by recombinant DNA techniques or genetic engineering.

In other embodiments, the polysaccharides are those from modifiedmicroalgae, such as, but not limited to, microalgae modified byrecombinant techniques. Non-limiting examples of such techniques includeintroduction and/or expression of an exogenous nucleic acid sequenceencoding a gene product; genetic manipulation to decrease or inhibitexpression of an endogenous microalgal gene product; and/or geneticmanipulation to increase expression of an endogenous microalgal geneproduct. The invention contemplates recombinant modification of thevarious microalgae species described herein. In some embodiments, themicroalgae is from the genus Porphyridium.

Polysaccharides provided by the invention that are produced bygenetically modified microalgae or microalgae that are provided with anexogenous carbon source can be distinct from those produced bymicroalgae cultured in minimal growth media under photoautotrophicconditions (ie: in the absence of a fixed carbon source) at least inthat they contain a different monosaccharide content relative topolysaccharides from unmodified microalgae or microalgae cultured inminimal growth media under photoautotrophic conditions. Non-limitingexamples include polysaccharides having an increased amount of arabinose(Ara), rhamnose (Rha), fucose (Fuc), xylose (Xyl), glucuronic acid(GlcA), galacturonic acid (GalA), mannose (Man), galactose (Gal),glucose (Glc), N-acetyl galactosamine (GalNAc), N-acetyl glucosamine(GlcNAc), and/or N-acetyl neuraminic acid (NANA), per unit mass (or permole) of polysaccharide, relative to polysaccharides from eithernon-genetically modified microalgae or microalgae culturedphotoautotrophically. An increased amount of a monosaccharide may alsobe expressed in terms of an increase relative to other monosaccharidesrather than relative to the unit mass, or mole, of polysaccharide. Anexample of genetic modification leading to production of modifiedpolysaccharides is transforming a microalgae with a carbohydratetransporter gene, and culturing a transformant in the presence of amonosaccharide which is transported into the cell from the culture mediaby the carbohydrate transporter protein encoded by the carbohydratetransporter gene. In some instances the culture can be in the dark,where the monosaccharide, such as glucose, is used as the sole energysource for the cell. In other instances the culture is in the light,where the cells undergo photosynthesis and therefore producemonosaccharides such as glucose in the chloroplast and transport themonosaccharides into the cytoplasm, while additional exogenouslyprovided monosaccharides are transported into the cell by thecarbohydrate transporter protein. In both instances monosaccharides fromthe cytoplasm are transported into the endoplasmic reticulum, wherepolysaccharide synthesis occurs. Novel polysaccharides produced bynon-genetically engineered microalgae can therefore be generated bynutritional manipulation, ie: exogenously providing carbohydrates in theculture media that are taken up through endogenous transport mechanisms.Uptake of the exogenously provided carbohydrates can be induced, forexample, by culturing the cells in the dark, thereby forcing the cellsto utilize the exogenously provided carbon source. For example,Porphyridium cells cultured in the presence of 7% glycerol in the darkproduce a novel polysaccharide because the intracellular carbon fluxunder these nutritionally manipulated conditions is different from thatunder photosynthetic conditions. Insertion of carbohydrate transportergenes into microalgae facilitates, but is not strictly necessary for,polysaccharide structure manipulation because expression of such genescan significantly increase the concentration of a particularmonosaccharide in the cytoplasm of the cell. Many carbohydratetransporter genes encode proteins that transport more than onemonosaccharide, albeit with different affinities for differentmonosaccharides (see for example Biochimica et Biophysica Acta 1465(2000) 263-274). In some instances a microalgae species can betransformed with a carbohydrate transporter gene and placed underdifferent nutritional conditions, wherein one set of conditions includesthe presence of exogenously provided galactose, and the other set ofconditions includes the presence of exogenously provided xylose, and thetransgenic species produces structurally distinct polysaccharides underthe two conditions. By altering the identity and concentration ofmonosaccharides in the cytoplasm of the microalgae, through geneticand/or nutritional manipulation, the invention provides novelpolysaccharides. Nutritional manipulation can also be performed, forexample, by culturing the microalgae in the presence of high amounts ofsulfate, as described herein. In some instances nutritional manipulationincludes addition of one or more exogenously provided carbon sources aswell as one or more other non-carbohydrate culture component, such as 50mM MgSO₄.

In some embodiments, the increase in one or more of the above listedmonosaccharides in a polysaccharide may be from below to abovedetectable levels and/or by at least about 5%, to at least about 2000%,relative to a polysaccharide produced from the same microalgae in theabsence of genetic or nutritional manipulation. Therefore an increase inone or more of the above monosaccharides, or other carbohydrates listedin Tables 2 or 3, by at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 100%, at least about 105%,at least about 110%, at least about 150%, at least about 200%, at leastabout 250%, at least about 300%, at least about 350%, at least about400%, at least about 450%, at least about 500%, at least about 550%, atleast about 600%, at least about 650%, at least about 700%, at leastabout 750%, at least about 800%, at least about 850%, at least about900%, at least about 1000%, at least about 1100%, at least about 1200%,at least about 1300%, at least about 1400%, at least about 1500%, atleast about 1600%, at least about 1700%, at least about 1800%, or atleast about 1900%, or more, may be used in the practice of theinvention.

In cases wherein the polysaccharides from unmodified microalgae do notcontain one or more of the above monosaccharides, the presence of themonosaccharide in a microalgal polysaccharide indicates the presence ofa polysaccharide distinct from that in unmodified microalgae. Thus,using particular strains of Porphyridium sp. and Porphyridium cruentumas non-limiting examples, the invention includes modification of thesemicroalgae to incorporate arabinose and/or facose, becausepolysaccharides from two strains of these species do not containdetectable amounts of these monosaccharides (see Example 5 herein). Inanother non-limiting example, the modification of Porphyridium sp. toproduce polysaccharides containing a detectable amount of glucuronicacid, galacturonic acid, or N-acetyl galactosamine, or more than a traceamount of N-acetyl glucosamine, is specifically included in the instantdisclosure. In a further non-limiting example, the modification ofPorphyridium cruentum to produce polysaccharides containing a detectableamount of rhamnose, mannose, or N-acetyl neuraminic acid, or more than atrace amount of N-acetyl-glucosamine, is also specifically included inthe instant disclosure.

Put more generally, the invention includes a method of producing apolysaccharide comprising culturing a microalgae cell in the presence ofat least about 0.01 micromolar of an exogenously provided fixed carboncompound, wherein the compound is incorporated into the polysaccharideproduced by the cell. In some embodiments, the compound is selected fromTable 2 or 3. The cells may optionally be selected from the specieslisted in Tables 1A or 1B, and cultured by modification, using themethods disclosed herein, or the culture conditions also listed in Table1A.

The methods may also be considered a method of producing a glycopolymerby culturing a transgenic microalgal cell in the presence of at leastone monosaccharide, wherein the monosaccharide is transported by thetransporter into the cell and is incorporated into a microalgalpolysaccharide.

In some embodiments, the cell is selected from Table 1A or 1B, such aswhere the cell is of the genus Porphyridium, as a non-limiting example.In some cases, the cell is selected from Porphyridium sp. andPorphyridium cruentum. Embodiments include those wherein thepolysaccharide is enriched for the at least one monosaccharide comparedto an endogenous polysaccharide produced by a non-transgenic cell of thesame species. The monosaccharide may be selected from Arabinose,Fructose, Galactose, Glucose, Mannose, Xylose, Glucuronic acid,Glucosamine, Galactosamine, Rhamnose and N-acetyl glucosamine.

These methods of the invention are facilitated by use of non-transgeniccell expressing a sugar transporter, optionally wherein the transporterhas a lower K_(m) for glucose than at least one monosaccharide selectedfrom the group consisting of galactose, xylose, glucuronic acid,mannose, and rhamnose. In other embodiments, the transporter has a lowerK_(m) for galactose than at least one monosaccharide selected from thegroup consisting of glucose, xylose, glucuronic acid, mannose, andrhamnose. In additional embodiments, the transporter has a lower K_(m)for xylose than at least one monosaccharide selected from the groupconsisting of glucose, galactose, glucuronic acid, mannose, andrhamnose. In further embodiments, the transporter has a lower K_(m) forglucuronic acid than at least one monosaccharide selected from the groupconsisting of glucose, galactose, xylose, mannose, and rhamnose. In yetadditional embodiments, the transporter has a lower K_(m) for mannosethan at least one monosaccharide selected from the group consisting ofglucose, galactose, xylose, glucuronic acid, and rhamnose. In yetfurther embodiments, the transporter has a lower K_(m) for rhamnose thanat least one monosaccharide selected from the group consisting ofglucose, galactose, xylose, glucuronic acid, and mannose. Manipulationof the concentration and identity of monosaccharides provided in theculture media, combined with use of transporters that have a differentK_(m) for different monosaccharides, provides novel polysaccharides.These general methods can also be used in cells other than microalgae,for example, bacteria that produce polysaccharides.

In alternative embodiments, the cell is cultured in the presence of atleast two monosaccharides, both of which are transporter by thetransporter. In some cases, the two monosaccharides are any two selectedfrom glucose, galactose, xylose, glucuronic acid, rhamnose and mannose.

In one non-limiting example, the method comprises providing a transgeniccell containing a recombinant gene encoding a monosaccharidetransporter; and culturing the cell in the presence of at least onemonosaccharide, wherein the monosaccharide is transported by thetransporter into the cell and is incorporated into a polysaccharide ofthe cell. It is pointed out that transportation of a monosaccharide fromthe media into a microalgal cell allows for the monosaccharide to beused as an energy source, as disclosed below, and for the monosaccharideto be transported into the endoplasmic reticulum (ER) by cellulartransporters. In the ER, polysaccharide production and glycosylationoccurs such that in the presence of exogenously providedmonosaccharides, the sugar content of the microalgal polysaccharideschange.

In some aspects, the invention includes a novel microalgalpolysaccharide, such as from microalgae of the genus Porphyridium,comprising detectable amounts of xylose, glucose, and galactose whereinthe molar amount of one or more of these three monosaccharides in thepolysaccharide is not present in a polysaccharide of Porphyridium thatis not genetically or nutritionally modified. An example of anon-nutritionally and non-genetically modified Porphyridiumpolysaccharide can be found, for example, in Jones R., Journal ofCellular Comparative Physiology 60; 61-64 (1962). In some embodiments,the amount of glucose, in the polysaccharide, is at least about 65% ofthe molar amount of galactose in the same polysaccharide. In otherembodiments, glucose is at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 100%, at least about 105%, at least about 110%, at leastabout 120%, at least about 130%, at least about 140%, at least about150%, at least about 200%, at least about 250%, at least about 300%, atleast about 350%, at least about 400%, at least about 450%, at leastabout 500%, or more, of the molar amount of galactose in thepolysaccharide. Further embodiments of the invention include thosewherein the amount of glucose in a microalgal polysaccharide is equalto, or approximately equal to, the amount of galactose (such that theamount of glucose is about 100% of the amount of galactose). Moreover,the invention includes microalgal polysaccharides wherein the amount ofglucose is more than the amount of galactose.

Alternatively, the amount of glucose in the polysaccharide is less thanabout 65% of the molar amount of galactose in the same polysaccharide.The invention thus provides for polysaccharides wherein the amount ofglucose is less than about 60%, less than about 55%, less than about50%, less than about 45%, less than about 40%, less than about 35%, lessthan about 30%, less than about 25%, less than about 20%, less thanabout 15%, less than about 10%, or less than about 5% of the molaramount of galactose in the polysaccharide.

In other aspects, the invention includes a microalgal polysaccharide,such as from microalgae of the genus Porphyridium, comprising detectableamounts of xylose, glucose, galactose, mannose, and rhamnose, whereinthe molar amount of one or more of these five monosaccharides in thepolysaccharide is not present in a polysaccharide of non-geneticallymodified and/or non-nutritionally modified microalgae. In someembodiments, the amount of rhamnose in the polysaccharide is at leastabout 100% of the molar amount of mannose in the same polysaccharide. Inother embodiments, rhamnose is at least about 110%, at least about 120%,at least about 130%, at least about 140%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 450%, or at least about 500%,or more, of the molar amount of mannose in the polysaccharide. Furtherembodiments of the invention include those wherein the amount ofrhamnose in a microalgal polysaccharide is more than the amount ofmannose on a molar basis.

Alternatively, the amount of rhamnose in the polysaccharide is less thanabout 75% of the molar amount of mannose in the same polysaccharide. Theinvention thus provides for polysaccharides wherein the amount ofrhamnose is less than about 70%, less than about 65%, less than about60%, less than about 55%, less than about 50%, less than about 45%, lessthan about 40%, less than about 35%, less than about 30%, less thanabout 25%, less than about 20%, less than about 15%, less than about10%, or less than about 5% of the molar amount of mannose in thepolysaccharide.

In additional aspects, the invention includes a microalgalpolysaccharide, such as from microalgae of the genus Porphyridium,comprising detectable amounts of xylose, glucose, galactose, mannose,and rhamnose, wherein the amount of mannose, in the polysaccharide, isat least about 130% of the molar amount of rhamnose in the samepolysaccharide. In other embodiments, mannose is at least about 140%, atleast about 150%, at least about 200%, at least about 250%, at leastabout 300%, at least about 350%, at least about 400%, at least about450%, or at least about 500%, or more, of the molar amount of rhamnosein the polysaccharide.

Alternatively, the amount of mannose in the polysaccharide is equal toor less than the molar amount of rhamnose in the same polysaccharide.The invention thus provides for polysaccharides wherein the amount ofmannose is less than about 95%, less than about 90%, less than about85%, less than about 80%, less than about 75%, less than about 70%, lessthan about 65%, less than about 60%, less than about 60%, less thanabout 55%, less than about 50%, less than about 45%, less than about40%, less than about 35%, less than about 30%, less than about 25%, lessthan about 20%, less than about 15%, less than about 10%, or less thanabout 5% of the molar amount of rhamnose in the polysaccharide.

In further aspects, the invention includes a microalgal polysaccharide,such as from microalgae of the genus Porphyridium, comprising detectableamounts of xylose, glucose, and galactose, wherein the amount ofgalactose in the polysaccharide, is at least about 100% of the molaramount of xylose in the same polysaccharide. In other embodiments,rhamnose is at least about 105%, at least about 110%, at least about120%, at least about 130%, at least about 140%, at least about 150%, atleast about 200%, at least about 250%, at least about 300%, at leastabout 350%, at least about 400%, at least about 450%, or at least about500%, or more, of the molar amount of mannose in the polysaccharide.Further embodiments of the invention include those wherein the amount ofgalactose in a microalgal polysaccharide is more than the amount ofxylose on a molar basis.

Alternatively, the amount of galactose in the polysaccharide is lessthan about 55% of the molar amount of xylose in the same polysaccharide.The invention thus provides for polysaccharides wherein the amount ofgalactose is less than about 50%, less than about 45%, less than about40%, less than about 35%, less than about 30%, less than about 25%, lessthan about 20%, less than about 15%, less than about 10%, or less thanabout 5% of the molar amount of xylose in the polysaccharide.

In yet additional aspects, the invention includes a microalgalpolysaccharide, such as from microalgae of the genus Porphyridium,comprising detectable amounts of xylose, glucose, glucuronic acid andgalactose, wherein the molar amount of one or more of these fivemonosaccharides in the polysaccharide is not present in a polysaccharideof unmodified microalgae. In some embodiments, the amount of glucuronicacid, in the polysaccharide, is at least about 50% of the molar amountof glucose in the same polysaccharide. In other embodiments, glucuronicacid is at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 100%, atleast about 105%, at least about 110%, at least about 120%, at leastabout 130%, at least about 140%, at least about 150%, at least about200%, at least about 250%, at least about 300%, at least about 350%, atleast about 400%, at least about 450%, or at least about 500%, or more,of the molar amount of glucose in the polysaccharide. Furtherembodiments of the invention include those wherein the amount ofglucuronic acid in a microalgal polysaccharide is more than the amountof glucose on a molar basis.

In other embodiments, the exopolysaccharide, or cell homogenatepolysaccharide, comprises glucose and galactose wherein the molar amountof glucose in the exopolysaccharide, or cell homogenate polysaccharide,is at least about 55% of the molar amount of galactose in theexopolysaccharide or polysaccharide. Alternatively, the molar amount ofglucose in the exopolysaccharide, or cell homogenate polysaccharide, isat least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,or at least about 100% of the molar amount of galactose in theexopolysaccharide or polysaccharide.

In yet further aspects, the invention includes a microalgalpolysaccharide, such as from microalgae of the genus Porphyridium,comprising detectable amounts of xylose, glucose, glucuronic acid,galactose, at least one monosaccharide selected from arabinose, fucose,N-acetyl galactosamine, and N-acetyl neuraminic acid, or any combinationof two or more of these four monosaccharides.

In one embodiment, a method of determining the amount of phycoerythrinper dry gram of cells in a formulated skin care composition is toquantify the amount of certain molecules known to be present at certainlevels or ranges of levels in Porphyridium cells. Such measurement is anindication of how many grams of dry Porphyridium biomass per gram offormulated skin care product are in a given formulation. For example,compounds listed in FIG. 7 are known to be present in Porphyridium cellsat certain ranges of levels. The amounts of compounds, such as thoselisted in FIG. 7, were determined by analysis of Porphyridium cellbiomass grown in nitrogen-replete media under outdoor daylightconditions, i.e., deep red cells containing quantities of phycoerythrintypically seen in cells grown in artificial seawater media such as thosedescribed in Example 1. Given a certain quantity of a compound in aformulated skin care composition, the skilled artisan can determine thenumber of dry grams of Porphyridium biomass are present per gram offormulated skin care product. The number of dry grams of Porphyridiumcells present in the composition can then be used to determine if thecells contain less than or more than a certain amount of phycoerythrinper dry gram of cells.

V. Cosmeceutical Compositions and Topical Application A. General

Compositions, comprising polysaccharides, whole cell extracts, ormixtures of polysaccharides and whole cell extracts, are provided fortopical application or non-systemic administration. The polysaccharidemay be any one or more of the microalgal polysaccharides disclosedherein, including those produced by a species, or a combination of twoor more species, in Tables 1A or 1B. Similarly, a whole cell extract maybe that prepared from a microalgal species, or a combination of two ormore species, in Tables 1A or 1B. In some embodiments, polysaccharides,such as exopolysaccharides, and cell extracts from microalgae of thegenus Porphyridium are used in the practice of the invention. Acomposition of the invention may comprise from between about 0.001% andabout 100%, about 0.01% and about 90%, about 0.1% and about 80%, about1% and about 70%, about 2% and about 60%, about 4% and about 50%, about6% and about 40%, about 7% and about 30%, about 8% and about 20%, orabout 10% polysaccharide, and/or cell extract, by weight.

In other embodiments, the composition comprises a carrier suitable fortopical administration and/or a preservative suitable for topicaladministration; and Rhodophyte cells, optionally of the genusPorphyridium. The cells may contain reduced amounts of the redpigmentation by preparation methods described herein. In some cases, anaqueous extract of a composition comprising the Porphyridium cellscontains no more than 75% to no more than about 1% of the absorbance pergram at 545 nm of a second composition formulated in identical fashionexcept containing cells of the same species of Porphyridium cells thatwere grown in a photobioreactor in ATCC 1495 ASW media in the presenceof 50 microeinsteins of light per square meter per second. In furtherembodiments, the carrier is suitable for topical administration tohumans, such as to human skin or a skin tissue.

In alternative embodiments, a composition for application to human skinmay comprise a polysaccharide isolated from cells of the genusPorphyridium. Such a composition may further comprise a carrier and/orpreservative suitable for topical administration as described herein. Insome cases, the polysaccharide of the composition contains no more thanabout 10% protein by weight. In other embodiments, the polysaccharidecontains no more than about 5%, no more than about 2%, or no more thanabout 1% protein by weight. The polysaccharide may also be essentially,or completely, free of protein, as detectable by assay methods asdescribed herein after treatment to remove protein.

In further embodiments, the polysaccharide may comprise a molar amountof glucose that is at least about 50%, or at least about 60%, of themolar amount of galactose. Alternatively, the molar amount of glucose inthe polysaccharide is greater than the molar amount of galactose. Inadditional embodiments, the polysaccharide contains less than a 0.1%, orless than a 0.01%, molar amount of at least one monosaccharide selectedfrom the group consisting of arabinose, rhamnose, fucose, and N-acetylglucosamine. Optionally, the polysaccharide contains less than a 0.1%,or less than a 0.01%, molar amount of each of arabinose, rhamnose,fucose, and N-acetyl glucosamine.

In yet additional embodiments, a composition may comprise apolysaccharide comprising a molar amount of glucose that is at leastabout 30%, or at least about 40%, of the molar amount of xylose. In somecases, the polysaccharide comprises a molar amount of glucose betweenabout 15.8% and about 25.8%; and a molar amount of xylose that isbetween about 37.5% and about 45.5%. Alternatively, the polysaccharidecomprises a molar amount of glucose between about 17.8% and about 23.8%;and a molar amount of xylose that is between about 39.5% and about43.5%.

In yet further embodiments, the polysaccharides are sulfatedexopolysaccharides containing at least about 3.0% sulfur, at least about3.5% sulfur, at least about 4.0% sulfur, at least about 4.5% sulfur, atleast about 4.6% sulfur, at least about 4.75% sulfur, at least about5.0% sulfur, at least about 5.25% sulfur, at least about 5.5% sulfur, atleast about 5.75% sulfur, at least about 6.0% sulfur, at least about6.25% sulfur, at least about 6.5% sulfur, at least about 6.75% sulfur,or at least about 7.0% sulfur by weight of the polysaccharide. Theamount or level of sulfation in the polysaccharides may be analyzed andcompared to the amount of sulfates used to culture the microalgae. Thus,the amount or level of sulfation in the polysaccharides of cells grownat about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM,about 600 mM, or about 700 mM or higher, sulfate (SO₄ ²⁻) may bedetermined by routine and repetitive methods disclosed herein. Theamount or level of sulfur by weight in the polysaccharides of a sampleof cells or cell material may be determined without knowledge of theamount of sulfate used to culture the cells.

As a further alternative, a composition for topical application to humanskin may comprise microalgal cells. The cells may be those of genusPorphyridium or any other species or strain as disclosed herein.Optionally, the composition further comprises a carrier and/orpreservative suitable for topical administration as described herein. Inalternative embodiments, the cells are homogenized (such as by methodsdescribed herein) to generate or form a microalgal cell homogenate. Insome cases, the cells or homogenate thereof, and therefore thecomposition, is essentially free of red and/or green coloration.Optionally, the cells or homogenate thereof, and therefore thecomposition, is completely free of red coloration. Thus, in someembodiments, the cells (and therefore the homogenate thereof) containsless than about 15, less than about 10, less than about 5, less thanabout 2, less than about 1, less than about 0.5, or less than about 0.1milligrams of phycoerythrin per dry gram of cells.

Alternatively, the cells (and therefore a homogenate thereof) contains asulfated polysaccharide having an amount of sulfur by weight of at least3.0%, at least 3.5%, at least 4.0%, at least 4.5%, at least 4.6%, atleast 4.75%, at least 5.0%, at least 5.25%, at least 5.5%, at least5.75%, at least 6.0%, at least 6.25%, at least 6.5%, at least 6.75%, orat least 7.0% sulfur by weight of the polysaccharide as describedherein. In additional embodiments, the microalgal cell homogenatecontains at least two, at least three, at least five, at least ten, orat least twenty times the amount of solvent-available polysaccharidepresent in a quantity of unhomogenized cells needed to generate themicroalgal cell homogenate.

In further embodiments, the disclosed invention includes a compositioncomprising particulate polysaccharides, such as microbeads or nanobeadscomprising a disclosed polysaccharide. In some embodiments thepolysaccharide particles are referred to as Marine Nanobeads™. Thecomposition may be for improving the appearance of skin, such as humanskin. The polysaccharides may have any level of sulfation describedherein. The composition may be sterile and/or non-pyrogenic andoptionally substantially free of endotoxins and/or proteins. In otherembodiments, the composition further comprises hyaluronic acid oranother agent suitable or desirable for treatment of skin. Non-limitingexamples of such an agent include aloe vera, urea, alpha hydroxyl acid,vitamin E, glycyrrhizinic acid, methylsulfonylmethane (MSM), andcollagen.

In some embodiments, the composition comprises an algal polysaccharide,wherein the polysaccharide: (a) has been made completely or partiallyinsoluble in water through drying; and (b) has been homogenized orotherwise milled or disrupted to generate particles.

The polysaccharide may of course be that of a variety of microalgalcells, such as those described in Tables 1A and 1B and those of thegenus Porphyridium. In some cases, the polysaccharide is contained in anon-aqueous material. As non-limiting examples, the material may becontained in an oil suitable for topical administration, withhexadecanoic acid or oil that is contained in an emulsion asrepresentative examples. In one embodiment, the particulatepolysaccharides from a species of the genus Porphyridium are formulatedin at least one of the following compounds: Ceraphyl (EthylhexylPalmitate), Aerosil (silica dimethyl silylate), butylene glycol, and apreservative such as diocide, dowicil 200, and methyl paraben. In someembodiments the concentrations of formulated particulate polysaccharidesfrom a species of the genus Porphyridium are between 50-97% Ceraphyl(Ethylhexyl Palmitate), 1-5% Aerosil (silica dimethyl silylate), 0.1-3butylene glycol, and 0.1-3% of a preservative such as diocide, dowicil200, and methyl paraben. The composition may also comprise a carrierand/or preservative suitable for topical administration. The compositionmay also be substantially free of endotoxins and/or protein as well assterile and/or non-pyrogenic. In further embodiments, the polysaccharideis encapsulated by a timed-release coating, such as one suitable fortopical application to human skin.

Optionally, the composition is prepared by a manufacturing orpreparation method as described herein, such as a method disclosed inthe following Methods of Formulation section. In some cases, thecomposition comprises a polysaccharide that is partially or completelyinsoluble in water, such as by heating an aqueous suspension of thepolysaccharide, thereby removing water from the suspension. Thepolysaccharide particulates may be partially soluble such that they areless than about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, less than about10%, less than about 5%, or less than about 2% soluble in water.

In other embodiments, the polysaccharide has been made partially orcompletely insoluble by a method selected from the group consisting ofchemical cross-linking, chemical dehydration through displacement ofbound water by an alcohol, precipitation from solution using an alcoholor a ketone or pH, and coating of particles by microencapsulation.Non-limiting examples of these methods are known to the skilled personand may be used in the practice of the invention. For examples, seeBiomacromolecules. 2005 November-December; 6(6):3202-8; ArteriosclerThromb Vasc Biol. 2004 March; 24(3):613-7; J Biomed Mater Res. 2001 Sep.15; 56(4): 478-86; Dalton Trans. 2004 Sep. 7; (17): 2621-34. Epub 2004Jul. 28; Biomacromolecules. 2004 January-February; 5(1): 126-36;Contraception. 2002 August; 66(2): 137-40; Biomacromolecules. 2006 May;7(5): 1471-80; Biopolymers. 1999 September; 50(3): 227-37; Biomaterials.2003 May; 24(12): 2083-96; Int J. Pharm. 2003 Nov. 28; 267(1-2): 13-25;Med Biol Eng Comput. 1998 January; 36(1):129-34 and Reprod Fertil Dev.2002; 14(5-6): 307-14. A representative example is chemicalcross-linking to a pharmaceutically or cosmetically acceptable insolublesolid phase material, such as a polymer, microbead, or nanobead. Theinsoluble material need not precipitate when in a solution but includesa material that remains in suspension when in solution. Dehydration orprecipitation with alcohol may be practiced with any alcohol suitablefor pharmaceutical or cosmetic use. Non-limiting examples includeethanol or a fatty alcohol such as cetyl, stearyl, cetearyl, or lanolinalcohol. A non-limiting method of microencapsulating a cosmetic isdescribed in U.S. Pat. No. 4,752,496.

The use of a disclosed method of the invention also includes milling ofdried polysaccharide material (such as a film) into particles by anysuitable method. Non-limiting examples of such methods are disclosedherein, and they produce particles with an average size that may rangebetween about 400 and about 0.1 microns.

In some embodiments, the composition comprises polysaccharide particlesthat increase in volume on contact with water compared to theiranhydrous or partially hydrated volume. In some embodiments, theparticles increase in volume by an amount selected from at least about5%, at least about 25%, at least about 50%, at least about 100%, atleast about 200%, at least about 300%, by at least about 500%, at leastabout 1000%, or at least about 5000%.

In some embodiments, the polysaccharide of the method is associated witha fusion protein as described herein. In some cases, the fusion proteincomprises a first protein with at least 60% amino acid identity with theprotein of SEQ ID NO: 15, and a second protein. Alternatively, thepolysaccharide of the method contains an amount of sulfur by weight fromat least about 3.0% sulfur to at least about 7.0% sulfur by weight asdescribed herein, and optionally is also associated with a fusionprotein. An example of an expression vector for expression inPorphyridium of a polysaccharide binding protein:superoxide dismutasefusion can be found in SEQ ID NO:36. In some embodiments a spacer of1-15 amino acids is placed between the glycoprotein and second proteinto enable flexibility between the two proteins. Expression of a fusionprotein, wherein the second heterologous protein is a dimerizing ormultimerizing protein (such as superoxide dismutase) can be advantageouswhen a higher viscosity or gelling property of the polysaccharide isdesired because the dimmers serve to crosslink the polysaccharide. Thereversibility of the crosslinking is in part dictated by the strength ofthe dimerization in such fusion proteins provided by the invention.

Topical compositions are usually formulated with a carrier, such as inan ointment or a cream, and may optionally include a fragrance. Onenon-limiting class of topical compositions is that of cosmeceuticals.Other non-limiting examples of topical formulations include gels,solutions, impregnated bandages, liposomes, or biodegradablemicrocapsules as well as lotions, sprays, aerosols, suspensions, dustingpowder, impregnated bandages and dressings, biodegradable polymers, andartificial skin. Another non-limiting example of a topical formulationis that of an ophthalmic preparation. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petroleum, white petroleum, propyleneglycol, polyoxyethylene polyoxypropylene compound, emulsifying wax andwater. Alternatively, the composition can be formulated with a suitablelotion or cream containing the active compound suspended or dissolved ina carrier. Suitable carriers include, but are not limited to, mineraloil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearylalcohol, 2-octyldodecanol, benzyl alcohol and water.

In some embodiments, the polysaccharides contain fucose moieties. Inother embodiments, the polysaccharides are sulfated, such asexopolysaccharides from microalgae of the genus Porphyridium. In someembodiments, the polysaccharides will be those from a Porphyridiumspecies, such as one that has been subject to genetic and/or nutritionalmanipulation to produce polysaccharides with altered monosaccharidecontent and/or altered sulfation.

In additional embodiments, a composition of the invention comprises amicroalgal cell homogenate and a topical carrier. In some embodiments,the homogenate may be that of a species listed in Table 1A or 1B or maybe material produced by a species in the tables.

In further embodiments, a composition comprising purified microalgalpolysaccharide and a carrier suitable for topical administration alsocontains a fusion (or chimeric) protein associated with thepolysaccharide. In some embodiments, the fusion protein comprises afirst protein, or polypeptide region, with at least about 60% amino acididentity with the protein of SEQ ID NO: 15. In other embodiments, thefirst protein has at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, or atleast about 98%, or higher, amino acid identity with the sequence of SEQID NO: 15. Preferably the fusion protein binds to a sulfatedexopolysaccharide from a cell of the genus Porphyridium. It ispreferable that the binding of the fusion protein to the polysaccharidebe selective and high affinity, though such is not required to practicethe invention.

The fusion protein may comprise a second protein, or polypeptide region,with a homogenous or heterologous sequence. Non-limiting examples of thesecond protein include an antibody, an enzyme, or a structural proteinof skin or a skin tissue, such as that of a human being. In some cases,the second protein is a peptide that binds to a surface on the exteriorof a human body. In various embodiments, the peptide can be selected tobind human skin, human hair, or human fingernails or toenails. Inoptional embodiments, the enzyme is superoxide dismutase, such as thathas at least about 60% amino acid identity with the sequence of SEQ IDNO:12, SEQ ID NO:13, or a protein from Table 21 and exhibit superoxidedismutase activity as non-limiting examples. In some embodiments, thesuperoxide dismutase has at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, or at least about 98%, or higher, amino acid identity with thesequence of SEQ ID NO: 12 or SEQ ID NO: 13. In other embodiments, thesecond protein is a structural skin protein selected from the groupconsisting of elastin and a collagen chain, such as that of human skin.Sequences encoding elastin and a chain of collagen are known to theskilled person and may be incorporated into a fusion protein via routinemethods. Examples of such human skin proteins are also disclosed hereinin the sequence listing. In one embodiment, the second protein iskeratin. Assays for superoxide dismutase activity are well known in theart. For examples see Song et al., Clin Sci (Lond). 2007 Jan. 8; Epubahead of print, Oxidative stress, antioxidant status and DNA damage inpatients with impaired glucose regulation and newly-diagnosed Type IIdiabetes; and Liu et al., Phytomedicine. 2006 Dec. 15 Protection of PC12cells from hydrogen peroxide-induced cytotoxicity by salvianolic acid B,a new compound isolated from Radix Salviae miltiorrhizae. The presenceof any exogenous or endogenous protein expressed in microalgae can beassayed for example, using well known methods such as western blottingand ELISA assays.

In other embodiments, the second protein is an antibody. Non-limitingexamples of antibodies for use in this aspect of the invention includean antibody that selectively binds to an antigen from a pathogenselected from HIV, Herpes Simplex Virus, gonorrhea, Chlamydia, HumanPapillomavirus, and Trichomoniasis. In some embodiments, the antibody isa humanized antibody.

B. Methods of Formulation

Polysaccharide compositions for topical application can be formulated byfirst preparing a purified preparation of polysaccharide. As anon-limiting example, the polysaccharide from aqueous growth media isprecipitated with an alcohol, resuspended in a dilute buffer, and mixedwith a carrier suitable for application to human skin or mucosal tissue,including the vaginal canal. Alternatively, the polysaccharide can bepurified from growth media and concentrated by tangential flowfiltration or other filtration methods, and formulated as describedabove. Intracellular polysaccharides can be also formulated in a similaror identical manner after purification from other cellular components.

As a non-limiting example, the invention includes a method offormulating a cosmeceutical composition, said method comprisingculturing microalgal cells in suspension under conditions to allow celldivision; separating the microalgal cells from culture media, whereinthe culture media contains exopolysaccharide molecules produced by themicroalgal cells; separating the exopolysaccharide molecules from othermolecules present in the culture media; homogenizing the microalgalcells; and adding the separated exopolysaccharide molecules to the cellsbefore, during, or after homogenization. In some embodiments, themicroalgal cells are from the genus Porphyridium.

In other embodiments, the invention includes a method of manufacturing acomposition comprising particles, the method comprising isolating apolysaccharide from microalgae; drying an aqueous suspension of thepolysaccharide to a solid film wherein at least some proportion of thefilm has been made completely or partially insoluble in water;homogenizing or otherwise milling or disrupting the film into particles;and formulating the particles into a non-aqueous material.

The method may of course be practiced with a variety of microalgalcells, such as those described in Tables 1A or 1B and those of the genusPorphyridium.

As described herein, the resulting composition may be for improving theappearance of skin, such as human skin. In some embodiments, theformulating may be into the oil phase of an oil-in-water emulsion. Inother embodiments, the non-aqueous material is an oil suitable fortopical administration, with hexadecanoic acid and oil that is containedin an emulsion as non-limiting examples. In further embodiments, themethod further comprises formulating the particles into a carrier and/orpreservative suitable for topical administration. The resultingcomposition may also be substantially free of endotoxins and/or protein.In many embodiments, the composition is also made sterile and/ornon-pyrogenic. Alternatively, the method further comprises formulatinghyaluronic acid into the composition.

In other embodiments, the polysaccharide after the drying step ispartially or completely insoluble in water. Optionally, thepolysaccharide after the drying step is soluble in water at a percentageselected from the list consisting of less than about 70%, less thanabout 60%, less than about 50%, less than about 40%, less than about30%, less than about 20%, less than about 10%, less than about 5%, andless than about 2%.

Embodiments of the drying step include drying performed at between about40 and about 180° C., such as between about 80° C. and about 170° C., orbetween about 100° C. and about 160° C., between about 125° C. and about155° C., between about 135° C. and about 152° C., between about 140° C.and about 150° C., or between about 145° C. and about 148° C. asnon-limiting examples. Optionally, the drying is performed in two steps,wherein the first step comprises heating the suspension of thepolysaccharide to no more than about 60° C. for a first period of timeto produce a solid film followed by heating the solid film for a secondperiod of time to no more than about 160° C. In alternative embodiments,the first and second steps comprise heating to no more than about 80° C.and no more than about 150° C., or to approximately 100° C. and no morethan 148° C., respectively. In some embodiments, the suspension of thepolysaccharide is heated during the first period of time in the presenceof air to produce a solid film and the solid film is heated during thesecond period of time in at least a partial vacuum or otherwise underreduced pressure.

After the drying step, milling may be by any suitable method.Non-limiting examples include a method selected from the list consistingof jet milling, ball milling, Retsch® milling, and milling in a Quadro®device. The resulting particles of the composition may have an averagesize between about 400 and about 0.1 microns. In some embodiments, theparticles of the composition have an average size between about 100 andabout 0.1 microns, between about 50 and about 0.1 microns, between about10 and about 0.1 microns, between about 10 and about 0.5 microns, orbetween about 5 and about 0.5 microns.

In some embodiments, the polysaccharide of the method is associated witha fusion protein as described herein. In some cases, the fusion proteincomprises a first protein with at least 60% amino acid identity with theprotein of SEQ ID NO: 15, and a second protein. Alternatively, thepolysaccharide of the method contains an amount of sulfur by weight fromat least about 3.0% sulfur to at least about 7.0% sulfur by weight asdescribed herein, and in some embodiments is associated with a fusionprotein.

Examples of polysaccharides, both secreted and intracellular, that aresuitable for formulation with a carrier for topical application arelisted in Table 1A.

In further embodiments, polysaccharide is associated with a fusion (orchimeric) protein comprising a first protein (or polypeptide region)with at least about 60% amino acid identity with the protein of SEQ IDNO: 15. In some cases, the first protein has at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 98%, or higher, amino acididentity with the sequence of SEQ ID NO:15.

The fusion protein may comprise a second protein, or polypeptide region,with a homogenous or heterologous sequence. One non-limiting example ofthe second protein is a superoxide dismutase enzyme.

Examples of carriers suitable for formulating polysaccharide aredescribed above. Ratios of homogenate:carrier are typically in the rangeof about 0.001:1 to about 1:1 (volume:volume), although the inventioncomprises ratios outside of this range, such as, but not limited to,about 0.01:1 and about 0.1:1.

Microalgal cellular extracts can also be formulated for topicaladministration. It is preferable but not necessary that the cells arephysically or chemically disrupted as part of the formulation process.For example, cells can be centrifuged from culture, washed with a buffersuch as 1.0 mM phosphate buffered saline, pH 7.4, and sonicated.Preferably the cells are sonicated until the cell walls have beensubstantially disrupted, as can be determined under a microscope. Forexample, Porphyridium sp. cells can be sonicated using a Misonixsonicator as described in Example 3.

Cells can also be dried and ground using means such as mortar andpestle, colloid milling, ball milling, or other physical method ofbreaking cell walls.

After cell disruption, cell homogenate can be formulated with carrierand fragrance as described above for polysaccharides.

The compositions according to the present invention can also be used ashair treating agents such as hair dressings (e.g., hair creams, hairsprays, hair tonics, hair gels, hair lotions, hair oils, hair essences,hair waters, hair waxes, and hair mousses), shampoos, finishing rinses,hair treatments, hair creams, hair mousses, hair setting lotions, haircolors, hair dyes (e.g., hair colors, one-part hair dyes, and two-parthair dyes), perm solutions (e.g., permanent wave solutions, hairstraightening solutions, and permanent wave holding agents), blood flowenhancers, scalp lotions, and anti-hair loss agents. Other applicationsof the compositions according to the present invention include, forexample, skin care cosmetics such as toners, serums, whitening toners,milky lotions, whitening milky lotions, creams, whitening creams,ointments, whitening ointments, lotions, whitening lotions, oils, andfacial packs. Furthermore, still other applications of the compositionsaccording to the present invention include, for example, makeupcosmetics such as foundations, liquid foundations, lipsticks, lipglosses, moisturizing lip balms, eye shadows, powders, face powders,blushers, eye shadows, eye liners, mascaras, and eyebrow pencils. Otherapplications of the compositions according to the present inventioninclude, for example, skin cleaners such as soap, cleansing creams,cleansing lotions, cleansing milks, cleansing pads, cosmeticcompositions, facial washes, and body shampoos. Moreover, anotherapplication of the compositions according to the present invention is infinishing cosmetics for use in, e.g., manicures. Other applications ofthe compositions according to the present invention include, forexample, cosmetic compositions in the form of bath agents, patches,perfumes, toothpastes, tooth washes, and mouthwashes.

Compositions in accordance with the present invention include finishedcosmetic products for oral or topical administration. Combinations ofvarious agents suitable or desirable for treatment of skin can beincluded in such finished cosmetic products. For example, compositionsfor topical administration can include algal polysaccharides, algalcarotenoids, vitamins (e.g., vitamin C, vitamin E, vitamin D, or vitaminD precursors), salicylic acid, amino acids (e.g., tyrosine), amino acidderivatives (e.g., N-acetyl methionine), betaine, perfluoropolyethers,whole cell algae extracts, dihydroxyacetone, Rhodiola rosea, menthol,and tissue respiratory factor. Compositions for oral administration caninclude, for example, fish oils, algal oils, vitamins (e.g., vitamin C,vitamin D, vitamin E), carotenoids, asthaxanthin, zeoxanthin, aminoacids (e.g., tyrosine), Rhodiola rosea, whole cell algal extracts, andbetaine. These components can be combined with suitable carriers, aswell as essential oils, fragrance oils, flavor oils, seed oils,botanicals, plant extracts, CO₂ extracts, soaps, clays, colorants,titanium dioxide, micas, tinting herbs, glitters, exfoliants, fruitseeds, fibers, grain powders, nut meals, seed meals, oil beads, waxbeads, herbs, hydrosols, vitamins, milk powders, preservatives,antioxidants, tocopherols, salts, sugars, vegetable oils, waxes,glycerin, sea vegetables, nutritive oils, moisturizing oils, vegetablebutters, propylene glycol, parabens, honey, bees wax, aloe, polysorbate,cornstarch, cocoa powder, coral powder, humectants, gums, emulsifyingagents, and/or thickeners.

In some embodiments, compositions in accordance with the presentinvention include algal polysaccharides in combination with at least oneother ingredient selected from the group consisting of beta carotene,lutein, astaxanthin, vitamin C, vitamin E, vitamin A, coenzyme Q10, apeptide, an aceylated peptide, oil soluble α-hydroxy acid, an alkyllactate, and salicylic acid. The polysaccharides and other ingredientscan be combined and subjected to processing (e.g., drying, heating,micronization, milling, and the like) to form particles comprising themultiple components for use in cosmetic, skin care, nutraceutical, orother products.

In one aspect, the invention is directed to cosmetic ingredients,including algal polysaccharides encapsulated in microspheres and aqueoussolutions of such microencapsulated algal polysaccharides. In someembodiments, the aqueous solutions can include from 0.1% to 10% w/wmicroencapsulated algal polysaccharides. The microspheres can beformulated to release the algal polysaccharide contents upon topicalapplication to, e.g., human skin, wherein the polysaccharides swell incontact with the skin's moisture to reduce the appearance of wrinkles onthe skin.

C. Co-Administered Compositions

Topical compositions can comprise a portion of a complete compositionsold as a single unit. Other portions of the complete compositions cancomprise an oral supplement intended for administration as part of aregime for altering skin appearance. Because the top layers of the skincontain dead cells, nutrients delivered via capillaries cannot reach theouter layers of cells. The outer layers of cells must be provided withnutrients though topical administration. However, topical administrationis not always an effective method of providing nutrients to deep layersof skin that contain living cells. The compositions provided hereincomprise both topical compositions that contain algal polysaccharidesand/or cellular extracts as well as oral compositions comprisingnutraceutical molecules such as purified polysaccharides, whole cellextracts, carotenoids, polyunsaturated fatty acids, and other moleculesthat are delivered to the skin via capillaries. The combined effect ofthe topical and oral administration of these molecules and extractsprovides a benefit to skin health that is additive or synergisticcompared to the use of only a topical or only an orally deliveredproduct.

Examples of the topical components of the composition includeexopolysaccharide from Porphyridium cruentum, Porphyridium sp., or othermicroalgae, e.g., those listed in Tables 1A and 1B. Other components ofthe topical composition can include polysaccharides and/or cell extractsfrom species listed in Table 1A and/or Table 1B.

Cellular extracts for topical administration can also include cellularhomogenates from microalgae that have been genetically engineered. Forexample, homogenates of Porphyridium sp. that have been engineered toexpress an exogenous gene encoding superoxide dismutase can beformulated for topical administration. Other genes that can be expressedinclude carotenoid biosynthesis enzymes and polyunsaturated fatty acidbiosynthesis enzymes.

Examples of compositions for oral administration include one or more ofthe following: DHA, EPA, ARA, lineoileic acid, lutein, lycopene, betacarotene, braunixanthin, zeaxanthin, astaxanthin, linoleic acid, alphacarotene, vitamin C and superoxide dismutase. Compositions for oraladministration usually include a carrier such as those described above.Oral compositions can be formulated in tablet or capsule form. Oralcompositions can also be formulated in an ingestible form such as afood, tea, liquid, etc. Oral compositions can, for example, comprise atleast 50 micrograms, at least 100 micrograms, at least 50 milligrams, atleast 100 milligrams, at least 500 milligrams, and at least one gram ofa small molecule such as a carotenoids or a polyunsaturated fatty acid.

In another aspect, the invention includes orally administerednutraceutical compositions comprising one or more polysaccharides, ormicroalgal cell extract or homogenate, of the invention. A nutraceuticalcomposition serves as a nutritional supplement upon consumption. Inother embodiments, a nutraceutical may be bioactive and serve to affect,alter, or regulate a bioactivity of an organism.

A nutraceutical may be in the form of a solid or liquid formulation. Insome embodiments, a solid formulation includes a capsule or tabletformulation as described above. In other embodiments, a solidnutraceutical may simply be a dried microalgal extract or homogenate, aswell as dried polysaccharides per se. In liquid formulations, theinvention includes suspensions, as well as aqueous solutions, ofpolysaccharides, extracts, or homogenates. In some embodiments thenutraceutical is derived from microalgae, while in other embodiments thenutraceutical is derived from other sources such as, for example,plants, plant extracts, and chemically synthesized molecules. In apreferred embodiment a topical composition and an oral compositioncontain at least one molecule in common.

The methods of the invention include a method of producing anutraceutical composition. Such a method may comprise drying amicroalgal cell homogenate or cell extract. The homogenate may beproduced by disruption of microalgae which has been separated fromculture media used to propagate (or culture) the microalgae. Thus in onenon-limiting example, a method of the invention comprises culturing redmicroalgae; separating the microalgae from culture media; disrupting themicroalgae to produce a homogenate; and drying the homogenate. Insimilar embodiments, a method of the invention may comprise drying oneor more polysaccharides produced by the microalgae.

In some embodiments, a method of the invention comprises drying by traydrying, spin drying, rotary drying, spin flash drying, orlyophilization. In other embodiments, methods of the invention comprisedisruption of microalgae by a method selected from pressure disruption,sonication, and ball milling

In additional embodiments, a method of the invention further comprisesformulation of the homogenate, extract, or polysaccharides with acarrier suitable for human consumption. As described herein, theformulation may be that of tableting or encapsulation of the homogenateor extract.

In further embodiments, the methods comprise the use of microalgalhomogenates, extracts, or polysaccharides wherein the cells contain anexogenous nucleic acid sequence, such as in the case of modified cellsdescribed herein. The exogenous sequence may encode a gene productcapable of being expressed in the cells or be a sequence which increasesexpression of one or more endogenous microalgal gene product.

In a preferred embodiment, at the topical composition and the oralcomposition both contain at least one molecule or one type of molecule(such as carotenoids) in common. For example, the topical compositioncontains homogenate of Porphyridium cells that contain zeaxanthin, andthe oral composition contains zeaxanthin. In another embodiment, thetopical composition contains homogenate of Porphyridium cells thatcontain polysaccharide, and the oral composition contains polysaccharidepurified from Porphyridium culture media. In another embodiment, thetopical composition and the oral composition both contain lutein and/orzeaxanthin. Microalgae can be but need not be the source of either orboth lutein and zeaxanthin. In some embodiments, the daily oralcomposition dosage is about 10 mg lutein and about 0.6 mg zeaxanthin perday, and the daily topical composition dosage is about 100 ppm luteinand about 12 ppm of zeaxanthin in an oil-free liquid. In otherembodiments the oral and topical compositions both comprise carotenoidssuch as at least one of phytoene, phytofluene, beta carotene, lutein,zeaxanthin, and astaxanthin.

In one embodiment, the topical composition, optionally derived orpartially derived from microalgae, comprises the carotenoids phytoeneand phytofluene, and optionally also contains vitamin C, and vitamin E;the oral composition, optionally derived or partially derived frommicroalgae, comprises at least two of the following compounds: ascorbicacid, vitamin E succinate, lutein, zeaxanthin, beta carotene, EPA, DHA,and CoQ10. All components of the oral composition do not necessarilyneed to be formulated into the same tablet or capsule. In somecompositions the oral composition comprises at least 3, at least 4, atleast 5, at least 6, and at least 7 of the aforementioned compounds.Optionally, daily dosages of the topical composition are about 0.1-7%IBR-CLC® concentrate (phytoene/phytofluene combination; IsraeliBiotechnology Research Corp.; U.S. Pat. No. 6,383,474), 1-20% vitamin C,1-20% vitamin E. Optionally, daily doses for the oral composition arebetween 20-600 mg ascorbic acid, 20-600 mg vitamin E succinate, 0.1-50mg lutein, 0.05-30 mg zeaxanthin, 0.1-50 mg beta carotene, 5-400 mgCoQ10, 50-750 mg EPA, and 20-750 mg DHA.

Some of the compositions described herein are packaged for sale as asingle unit. For example, a unit for sale comprises a first containerholding a composition for topical administration, a second containerholding individual doses of a composition for oral administration, andoptionally, directions for co-administration of the topical and oralcomposition.

Some embodiments of the invention include a combination productcomprising 1) a first composition comprising a microalgal extract and acarrier suitable for topical application to skin; and 2) a secondcomposition comprising at least one compound and a carrier suitable forhuman consumption; wherein the first and second compositions arepackaged for sale as a single unit. Thus the invention includesco-packaging of the two compositions, optionally with a instructionsand/or a label indicating the identity of the contents and/or theirproper use.

Other combination products are including in the invention. In someembodiments, the first composition may be a topical formulation ornon-systemic formulation, optionally a cosmeceutical, as describedherein. Preferably, the first composition comprises a carrier suitablefor topical application to skin, such as human skin. Non-limitingexamples of the second composition include a food composition ornutraceutical as described herein. Preferably, the second compositioncomprises at least one carrier suitable for human consumption, such asthat present in a food product or composition. Combination products ofthe invention may be packaged separately for subsequent use together bya user or packaged together to facilitate purchase and use by aconsumer. Packaging of the first and second compositions may be for saleas a single unit.

D. Methods of Cosmetic Enhancement

In a further aspect, the invention includes a method to cosmeticallyenhance skin or its appearance or texture. In some cases, theenhancement is due to increased or improved skin elasticity. The skinmay be that of a human being, such as the skin of the face, hands, feet,or other parts of the human body. In other embodiments, the enhancementmay be in the appearance or texture of human lips. The method maycomprise administration of a polysaccharide composition suitable forinjection into skin or lip tissue to improve the appearance thereof. Thecomposition may be any as described herein suitable for the method ofadministration or application. In some embodiments, the injection ismade to alleviate or eliminate wrinkles. In other embodiments, thetreatment reduces the visible signs of aging and/or wrinkles.

As known to the skilled person, human skin, as it ages, gradually losesskin components that keep skin pliant and youthful-looking. The skincomponents include collagen, elastin, and hyaluronic acid, which havebeen the subject of interest and use to improve the appearance of agingskin.

The invention includes compositions of microalgal polysaccharides,microalgal cell extracts, and microalgal cell homogenates for use in thesame manner as collagen and hyaluronic acid. In some embodiments, thepolysaccharides will be those of from a Porphyridium species, such asone that has been subject to genetic and/or nutritional manipulation toproduce polysaccharides with altered monosaccharide content and/oraltered sulfation. In some embodiments, the polysaccharides areformulated as a fluid, optionally elastic and/or viscous, suitable forinjection. The compositions may be used as injectable dermal fillers asone non-limiting example. The injections may be made into skin tofill-out facial lines and wrinkles. In other embodiments, the injectionsmay be used for lip enhancement. These applications of polysaccharidesare non-limiting examples of non-pharmacological therapeutic methods ofthe invention.

In further embodiments, the microalgal polysaccharides, cell extracts,and cell homogenates of the invention may be co-formulated with collagenand/or hyaluronic acid (such as the Restylane® and Hylaform® products)and injected into facial tissue. Non-limiting examples of such tissueinclude under the skin in areas of wrinkles and the lips. In a preferredembodiment, the polysaccharide is substantially free of protein. Theinjections may be repeated as deemed appropriate by the skilledpractitioner, such as with a periodicity of about three, about four,about six, about nine, or about twelve months. In another preferredembodiment, a hyaluronic acid material is mixed with a polysaccharidefrom the genus Porphyridium prior to co-administration. The invention inthis particular embodiment provides longer half-life to the hyaluronicacid due to the potent inhibition of hyaluronidase by polysaccharidesisolated from microalgae from the genus Porphyridium. This allows forless injections to a patient. Preferably the polysaccharide from thegenus Porphyridium is at least substantially free of protein. Preferablythe mixture of polysaccharide from the genus Porphyridium and hyaluronicacid is sterile.

Thus, the invention includes a method of cosmetic enhancement comprisinginjecting a polysaccharide produced by microalgae into mammalian skin.The injection may be of an effective amount to produce a cosmeticimprovement, such as decreased wrinkling or decreased appearance ofwrinkles as non-limiting examples. Alternatively, the injection may beof an amount which produces relief in combination with a series ofadditional injections. In some methods, the polysaccharide is producedby a microalgal species, or two or more species, listed in Tables 1A or1B. In one non-limiting example, the microalgal species is of the genusPorphyridium and the polysaccharide is substantially free of protein.

The invention further includes a method to inhibit hyaluronidaseactivity comprising contacting the hyaluronidase with a disclosedpolysaccharide. In some embodiments, the hyaluronidase activity is inthe skin or a skin tissue of a human subject and the contactingcomprises administering the polysaccharide to the subject. Theadministering may comprise injection of the polysaccharide, or apolysaccharide containing composition of the invention, to the skin orskin tissue and/or to the lips or a lip tissue. The amount ofpolysaccharide administered may be any that is sufficient or effectiveto inhibit hyaluronidase activity to a level as desired by a skilledperson. The level of reduction in hyaluronidase activity may bedetermined by routine methods, including a comparative method whereinthe activity in the presence of polysaccharide is compared to theactivity in the absence thereof. Thus the amount of reduction may be atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, or about 90% or higher than that observed in theabsence of polysaccharide. In a preferred embodiment the polysaccharideused to inhibit hyaluronidase is from a species of the genusPorphyridium.

The invention also includes a method to stimulate procollagen and/orcollagen synthesis or production in a cell, such as a human fibroblast,by contacting the cell with a disclosed polysaccharide. In someembodiments, the cell is in the skin of a human subject and thecontacting comprises administering the polysaccharide to the subject.The administering may comprise injection of the polysaccharide, or apolysaccharide containing composition of the invention, to the skin or askin tissue and/or to the lips or a lip tissue. The amount ofpolysaccharide administered may be any that is sufficient or effectiveto stimulate procollagen or collagen synthesis to a level desired by askilled person, such as an increase of at least about 5%, 10%, about20%, or about 30% or higher than that observed in the absence ofpolysaccharide. In a related manner, the polysaccharide may be used toinhibit collagenase activity. The inhibition may be sufficient to resultin an increase of procollagen or collagen levels as described above.

Additionally, the invention includes a method to stimulate elastinsynthesis or production in a cell, such as a fibroblast, by contactingthe cell with a disclosed polysaccharide. In a related manner, thepolysaccharide may also inhibit elastase activity produced by a cell,such as, but not limited to, a fibroblast. In some embodiments, the cellis in the skin of a human subject and the contacting comprisesadministering the polysaccharide to the subject. The administering maycomprise injection of the polysaccharide, or a polysaccharide containingcomposition of the invention, to the skin or a skin tissue. The amountof polysaccharide administered may be any that is sufficient oreffective to stimulate elastin synthesis to a level desired by a skilledperson, such as an increase of at least about 50%, 100%, about 200%, orabout 300% or higher than that observed in the absence ofpolysaccharide. In a preferred embodiment the polysaccharide stimulatingelastin secretion contains at least 5.0% sulfur by weight. Similarly,the polysaccharide may decrease elastase activity by about 10%, about20%, about 30%, about 40%, about 50%, or about 60% or higher than thatobserved in the absence of polysaccharide.

The invention further includes the use of the disclosed polysaccharidesbased on their observed anti-oxidant activity. Thus the inventionincludes a method of providing anti-oxidant activity to skin or a skintissue, such as that of a human subject, by administering apolysaccharide. In some embodiments, the method inhibits reactive oxygenspecies (ROS) formation and/or activity in the skin. The invention thusincludes a method to prevent or treat a disease or unwanted conditionassociated with ROS or oxidative stress. Non-limiting examples of such adisease or unwanted condition include reducing inflammation orirritation of human skin or lips. In some embodiments, thepolysaccharide composition comprises one or more other agents orcompounds with anti-oxidant activity. Non-limiting examples of otheragents include vitamin A (retinyl palmitate), vitamin C (such as one ormore of ascorbyl palmitate, sodium ascorbyl palmitate, andtetrahexyldecyl ascorbate), vitamin D (cholecalcipherol), vitamin E(such as tocopheryl acetate and tocopherol/D-alpha tocopherol), alphalipoic acid, coenzyme, L-selenomethionine, and beta glucan.

In a related manner, a polysaccharide is used based on itsanti-inflammatory in skin or a skin tissue. In some embodiments, themethod inhibits polymorphonuclear (PMN) leukocytes in chemotaxis, suchas to sites of inflammation in skin. The level of inhibition may beabout 10%, about 20%, about 30%, about 40%, or about 50% or more thanthat seen in the absence of polysaccharide. In other embodiments, themethod inhibits the synthesis or release of a pro-inflammatory cytokine,such as interferon-gamma or interleukin-1-alpha. With interferon-gammaas an example, the inhibition may be at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, orabout 90% or more than that observed in the absence of polysaccharide.With interleukin-1-alpha as an example, the inhibition may be at leastabout 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, or about 80% or more than that observed in the absence ofpolysaccharide. In further embodiments, the method inhibitsproliferation of peripheral blood mononuclear cells, includinglymphocytes, monocytes, and macrophages. The level of inhibition may beabout 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, or about 80% or more than that observed in the absence ofpolysaccharide.

The above described methods may be individually part of a method toreduce the signs of aging or reduce the appearance of aging in humanskin as described herein. The methods may also be based upon the insightthat the microalgal biomass and polysaccharides of the invention alsoreduce the effects of UV light or radiation. In some embodiments, thepolysaccharide reduces thymidine dimer formation in DNA caused byexposure to UVB irradiation. The reduction may be at least about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, orabout 80% or more than that observed in the absence of polysaccharide.

In a related manner, the disclosed methods can be used to shield humanskin or lip tissue from UV light radiation. The UV radiation maycomprise UVA and/or UVB. The method may comprise applying a compositionof the disclosed invention to skin or a skin tissue in an effective orsufficient amount to shield, at least in part, the skin from UVradiation. In some embodiments, the amount is that which reducesthymidine dimer formation and/or sunburn. In an alternative embodiment,a composition of the invention may be applied in an effective orsufficient amount, such as that which reduces further UV-mediateddamage, to treat skin that has been damaged by UV radiation. Anadditional non-limiting example is a method of for treating skin toreduce the risk of skin cancer induced by sunlight or UV radiation.

The polysaccharide compositions may be in the form of a sterile and/ornon-pyrogenic injectable preparation, for example, as a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to techniques known in the art using suitabledispersing or wetting agents (such as, for example, Tween 80) andsuspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that maybe employed are mannitol, water, Ringers solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose, any blandfixed oil may be employed including synthetic mono- or diglycerides.Fatty acids, such as oleic acid and its glyceride derivatives are usefulin the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersantsuch as Ph. Helv or a similar alcohol.

Sterile injectable polysaccharide compositions preferably contain lessthan 1% protein as a function of dry weight of the composition, morepreferably less than 0.1% protein, more preferably less than 0.01%protein, less than 0.001% protein, less than 0.0001% protein, morepreferably less than 0.00001% protein, more preferably less than0.000001% protein.

VI. Gene Expression in Microalgae

Genes can be expressed in microalgae by providing, for example, codingsequences in operable linkage with promoters.

An exemplary vector design for expression of a gene in microalgaecontains a first gene in operable linkage with a promoter active inalgae, the first gene encoding a protein that imparts resistance to anantibiotic or herbicide. Exemplary antibiotic resistance genes include,without limitation, a Porphyridium sp. sulfometuron resistance gene (SEQID NO:10), and a zeocin resistance gene (SEQ ID NO:24). Optionally, thefirst gene is followed by a 3′ untranslated sequence containing apolyadenylation signal. The vector may also contain a second promoteractive in algae in operable linkage with a second gene. The second genecan encode any protein, for example an enzyme that produces smallmolecules or a mammalian growth hormone that can be advantageouslypresent in a nutraceutical.

It is preferable to use codon-optimized cDNAs: for methods of recodinggenes for expression in microalgae, see for example US patentapplication 20040209256.

It has been shown that many promoters in expression vectors are activein algae, including both promoters that are endogenous to the algaebeing transformed algae as well as promoters that are not endogenous tothe algae being transformed (i.e., promoters from other algae, promotersfrom plants, and promoters from plant viruses or algae viruses).Suitable promoters include, without limitation, Chlamydomonasreinhardtii RBCS2 promoters (SEQ ID NO:1 and SEQ ID NO:16),Chlamydomonas reinhardtii β-2 tubulin promoter (SEQ ID NO:2), Chlorellavirus promoter #1 (SEQ ID NO:3), Chlorella virus promoter #2 (SEQ IDNO:4), Chlorella virus promoter #3 (SEQ ID NO:5), Rhodella reticulatephycoerythrin beta subunit promoter (SEQ ID NO:6), Porphyridium AHASpromoter (SEQ ID NO:7), Porphyridium purpureum carbonic anhydrase pca2promoter (SEQ ID NO:8), and Porphyridium purpureum carbonic anhydrasepca1 promoter (SEQ ID NO:9).

Exemplary methods for transforming microalgae, in addition to thosedemonstrated in the Examples section below, including methods comprisingthe use of exogenous and/or endogenous promoters that are active inmicroalgae, and antibiotic resistance genes functional in microalgae,have been described. See for example; Curr Microbiol. 1997 December;35(6):356-62 (Chlorella vulgaris); Mar Biotechnol (NY). 2002 January;4(1):63-73 (Chlorella ellipsoidea); Mol Gen Genet. 1996 Oct. 16; 252(5):572-9 (Phaeodactylum tricornutum); Plant Mol. Biol. 1996 April;31(1):1-12 (Volvox carteri); Proc Natl Acad Sci U S A. 1994 Nov. 22;91(24): 11562-6 (Volvox carteri); Falciatore A, Casotti R, Leblanc C,Abrescia C, Bowler C, PMID: 10383998, 1999 May; 1(3):239-251 (Laboratoryof Molecular Plant Biology, Stazione Zoologica, VIIIa Comunale, 1-80121Naples, Italy) (Phaeodactylum tricornutum and Thalassiosiraweissflogii); Plant Physiol. 2002 May; 129(1):7-12. (Porphyridium sp.);Proc Natl Acad Sci USA. 2003 Jan. 21; 100(2): 438-42. (Chlamydomonasreinhardtii); Proc Natl Acad Sci USA. 1990 February; 87(3):1228-32.(Chlamydomonas reinhardtii); Nucleic Acids Res. 1992 Jun. 25; 20(12):2959-65; Mar Biotechnol (NY). 2002 January; 4(1):63-73 (Chlorella);Biochem Mol Biol Int. 1995 August; 36(5): 1025-35 (Chlamydomonasreinhardtii); J Microbiol. 2005 August; 43(4):361-5 (Dunaliella); YiChuan Xue Bao. 2005 April; 32(4):424-33 (Dunaliella); Mar Biotechnol(NY). 1999 May; 1(3):239-251. (Thalassiosira and Phaedactylum);Koksharova, Appl Microbiol Biotechnol 2002 February; 58(2):123-37(various species); Mol Genet Genomics. 2004 February; 271(1):50-9(Thermosynechococcus elongates); J. Bacteriol. (2000), 182, 211-215;FEMS Microbiol Lett. 2003 Apr. 25; 221(2): 155-9; Plant Physiol. 1994June; 105(2):635-41; Plant Mol. Biol. 1995 December; 29(5):897-907(Synechococcus PCC 7942); Mar Pollut Bull. 2002; 45(1-12): 163-7(Anabaena PCC 7120); Proc Natl Acad Sci USA. 1984 March; 81(5):1561-5(Anabaena (various strains)); Proc Natl Acad Sci USA. 2001 Mar. 27;98(7): 4243-8 (Synechocystis); Wirth, Mol Gen Genet. 1989 March;216(1):175-7 (various species); Mol Microbiol, 2002 June; 44(6):1517-31and Plasmid, 1993 September; 30(2):90-105 (Fremyella diplosiphon); Hallet al. (1993) Gene 124: 75-81 (Chlamydomonas reinhardtii); Gruber et al.(1991). Current Micro. 22: 15-20; Jarvis et al. (1991) Current Genet.19: 317-322 (Chlorella); for additional promoters see also Table 1 fromU.S. Pat. No. 6,027,900).

Suitable promoters may be used to express a nucleic acid sequence inmicroalgae. In some embodiments, the sequence is that of an exogenousgene or nucleic acid. In some embodiments the exogenous gene can encodea superoxide dismutase (SOD) or an SOD fusion. In cases of an exogenousnucleic acid coding sequence, the codon usage may be optionallyoptimized in whole or in part to facilitate expression in microalgae.

In some embodiments the invention includes cells of the genusPorphyridium that have been stably transformed with a vector containinga selectable marker gene in operable linkage with a promoter active inmicroalgae. In other embodiments the invention includes cells of thegenus Porphyridium that have been stably transformed with a vectorcontaining a selectable marker gene in operable linkage with a promoterendogenous to a member of the Rhodophyte order. Such promoters includeSEQ ID NO:6, SEQ ID NO:20 and SEQ ID NO:21, promoters from the genome ofChondrus crispus (Genbank accession number Z47547), promoters from thegenome of Cyanidioschyzon merolae (see for example Matsuzaki, M. et al.Nature 428, 653-657 (2004); Plant Physiology 137: 567-585 (2005); entiresequence available athttp://merolae.biol.s.u-tokyo.ac.jp/db/chromosome.cgi). In otherembodiments the invention includes cells of the genus Porphyridium thathave been stably transformed with a vector containing a selectablemarker gene in operable linkage with a promoter other than a CMVpromoter such as that found in PCT application WO2006013572.

In other embodiments, the invention provides for the expression of aprotein sequence found to be tightly associated with microalgalpolysaccharides. One non-limiting example is the protein of SEQ ID NO:15, which has been shown to be tightly associated with, but notcovalently bound to, the polysaccharide from Porphyridium sp. (see J.Phycol. 40: 568-580 (2004)). When Porphyridium culture media issubjected to tangential flow filtration using a filter containing a poresize well in excess of the molecular weight of the protein of SEQ ID NO:15, the polysaccharide in the retentate contains detectable amounts ofthe protein, indicating its tight association with the polysaccharide.The calculated molecular weight of the protein is approximately 58 kD,however with glycosylation the protein is approximately 66 kD.

Such a protein may be expressed directly such that it will be presentwith the polysaccharides of the invention or expressed as part of afusion or chimeric protein as described herein. As a fusion protein, theportion that is tightly associated with a microalgal polysaccharideeffectively links the other portion(s) to the polysaccharide. A fusionprotein may comprise a second protein or polypeptide, with a homogenousor heterologous sequence. A homogenous sequence would result in a dimeror multimer of the protein while a heterologous sequence can introduce anew functionality, including that of a bioactive protein or polypeptide.

Non-limiting examples of the second protein include an enzyme. Inoptional embodiments, the enzyme is superoxide dismutase, such as thathas at least about 60% amino acid identity with the sequence of SEQ IDNO: 12, SEQ ID NO: 13, and proteins from Table 21 as non-limitingexamples. Exemplary fusion proteins include superoxide dismutase fusedto a polysaccharide binding protein (SEQ ID NO: 14 or SEQ ID NO: 17). Insome embodiments, the superoxide dismutase has at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 98%, or higher, amino acididentity with the sequence of SEQ ID NO:12 or SEQ ID NO:13 or a proteinfrom Table 21. In other embodiments, the enzyme is a phytase (such asGenBank accession number CAB91845 and U.S. Pat. Nos. 6,855,365 and6,110,719). Other examples of second proteins include structuralproteins from mammalian skin such as collagen and elastin. Exemplaryfusion proteins include a glycoprotein-collagen fusion (SEQ ID NO:22),and a glycoprotein-elastin fusion (SEQ ID NO:23). Assays such as wesytemblot and ELISAs can be used to confirm the presence of the secondprotein in the biomass as well as when it is attached to a purifiedpolysaccharide. Polysaccharides with fusion proteins bound can bepurified as in Example 2. Activity assays for proteins such as phytasesand superoxide dismutase are well known in the art.

One advantage to a fusion is that the bioactivity of the polysaccharideand the bioactivity from the protein can be combined in a productwithout increasing the manufacturing cost over only purifying thepolysaccharide. As a non-limiting example, the potent antioxidantproperties of a Porphyridium polysaccharide can be combined with thepotent antioxidant properties of superoxide dismutase in a fusion,however the polysaccharide:superoxide dismutase combination can beisolated to a high level of purity using tangential flow filtration. Inanother non-limiting example, the potent antiviral properties of aPorphyridium polysaccharide can be added to the potent neutralizingactivity of recombinant antibodies fused to the protein (SEQ ID NO: 15)that tightly associates with the polysaccharide.

Another preferred fusion is between a protein with 50-100% amino acididentity with SEQ ID NO: 15, which retains the ability to bind aPorphyridium polysaccharide, and a peptide that bind to an externalsurface on a human body, such as hair, skin, fingernails, or toenails.Nucleic acid sequences encoding such peptides are cloned in-frame withthe polysaccharide binding protein, such as at the C-terminus. In someembodiments a linker peptide can be placed between the skin-binding,hair-binding, or fingernail-binding peptide and the protein that bindsthe polysaccharide. An example of such a linker is SEQ ID NO:41, or asubsegment thereof. Flexible linker proteins are well known in the art.See Appl Microbiol Biotechnol (2002) 58: 658-662 for examples. In oneembodiment, the fusion protein contains a linker of 1-50 amino acidsbetween the first protein and the second protein.

Examples of peptides for use in the invention described herein that bindexterior surfaces of the human body can be found, for example, in U.S.Pat. Nos. 7,129,326 and 7,220,405. For example, the peptides LESTPKMKand LESTPKM (SEQ ID NO:42 and SEQ ID NO:43) bind tightly to human hairand thus serve to anchor polysaccharide from Porphyridium to human hair.The peptide FTQSLPP (SEQ ID NO:44) binds tightly to human skin and thusserves to anchor polysaccharide from Porphyridium to human skin. Thepeptide ALPRIANTWSPS (SEQ ID NO:45) binds tightly to human fingernailsand thus serves to anchor polysaccharide from Porphyridium to humanfingernails. An example of a skin-binding peptide fusion with a flexiblelinker between the skin-binding peptide and the protein that binds thePorphyridium polysaccharide is SEQ ID NO:46.

Preferred carbohydrate transporters for expression in Porphyridium areSEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:39,and SEQ ID NO:40.

In other embodiments, the invention includes genetic expression methodscomprising the use of an expression vector. In one method, a microalgalcell, such as a Porphyridium cell, is transformed with a dual expressionvector under conditions wherein vector mediated gene expression occurs.The expression vector may comprise a resistance cassette comprising agene encoding a protein that confers resistance to an antibiotic such aszeocin, operably linked to a promoter active in microalgae. The vectormay also comprise a second expression cassette comprising a secondprotein to a promoter active in microalgae. The two cassettes arephysically linked in the vector. The transformed cells may be optionallyselected based upon the ability to grow in the presence of theantibiotic or other selectable marker under conditions wherein cellslacking the resistance cassette would not grow, such as in the dark. Theresistance cassette, as well as the expression cassette, may be taken inwhole or in part from another vector molecule.

In one non-limiting example, a method of expressing an exogenous gene ina cell of the genus Porphyridium is provided. The method may compriseoperably linking a gene encoding a protein that confers resistance tothe antibiotic zeocin to a promoter active in microalgae to form aresistance cassette; operably linking a gene encoding a second proteinto a promoter active in microalgae to form a second expression cassette,wherein the resistance cassette and second expression cassette arephysically connected to form a dual expression vector; transforming thecell with the dual expression vector; and selecting for the ability tosurvive in the presence of at least 2.5 ug/ml zeocin, preferably atleast 3.0 ug/ml zeocin, and more preferably at least 3.5 ug/ml zeocin,more preferably at least 5.0 ug/ml zeocin.

In additional aspects, the expression of a protein that produces smallmolecules in microalgae is included and described. Some genes that canbe expressed using the methods provided herein encode enzymes thatproduce nutraceutical small molecules such as lutein, zeaxanthin, andDHA. Preferably the genes encoding the proteins are synthetic and arecreated using preferred codons on the microalgae in which the gene is tobe expressed. For example, enzyme capable of turning EPA into DHA arecloned into the microalgae Porphyridium sp. by recoding genes to adaptto Porphyridium sp. preferred codons. For examples of such enzymes seeNat. Biotechnol. 2005 August; 23(8):1013-7. For examples of enzymes inthe carotenoid pathway see SEQ ID NO:18 and SEQ ID NO:19 and Table 22.The advantage to expressing such genes is that the nutraceutical valueof the cells increases without increasing the manufacturing cost ofproducing the cells.

For sequence comparison to determine percent nucleotide or amino acididentity, typically one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra.). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. For identifying whether a nucleic acid or polypeptide is withinthe scope of the invention, the default parameters of the BLAST programsare suitable. The BLASTN program (for nucleotide sequences) uses asdefaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4,and a comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a word length (W) of 3, an expectation (E) of10, and the BLOSUM62 scoring matrix. The TBLATN program (using proteinsequence for nucleotide sequence) uses as defaults a word length (W) of3, an expectation (E) of 10, and a BLOSUM 62 scoring matrix. (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

It should be apparent to one skilled in the art that various embodimentsand modifications may be made to the invention disclosed in thisapplication without departing from the scope and spirit of theinvention. All publications mentioned herein are cited for the purposeof describing and disclosing reagents, methodologies and concepts thatmay be used in connection with the present invention. Nothing herein isto be construed as an admission that these references are prior art inrelation to the inventions described herein.

U.S. Pat. No. 7,135,290 is hereby incorporated by reference in itsentirety for all purposes. The following applications are alsoincorporated by reference in their entirety for all purposes: U.S.patent application Ser. No. 11/336,426, filed Jan. 19, 2006, entitled“Polysaccharide Compositions and Methods of Administering, Producing,and Formulating Polysaccharide Compositions”; U.S. patent applicationSer. No. 11/337,103, filed Jan. 19, 2006, entitled “Methods andCompositions for Improving the Health and Appearance of Skin”; U.S.patent application Ser. No. 11/336,656, filed Jan. 19, 2006, entitled“Devices and Solutions for Prevention of Sexually Transmitted Diseases”;U.S. patent application Ser. No. 11/336,428, filed Jan. 19, 2006,entitled “Methods and Compositions for Cholesterol Reduction inMammals”; U.S. patent application Ser. No. 11/337,171, filed Jan. 19,2006, entitled “Methods and Compositions for Reducing Inflammation andPreventing Oxidative Damage”; U.S. patent application Ser. No.11/336,431, filed Jan. 19, 2006, entitled “Methods and Compositions forThickening, Stabilizing and Emulsifying Foods”; U.S. patent applicationSer. No. 11/336,430, filed Jan. 19, 2006, entitled “Methods andCompositions for Joint Lubrication”; U.S. Patent Application No.60/832,091, filed Jul. 20, 2006, entitled “Decolorized MicroalgalCompositions for Skin Care Products”; U.S. Patent Application No.60/838,452, filed Aug. 17, 2006, entitled “Polysaccharide Compositionsand Methods of Administering, Producing, and Formulating PolysaccharideCompositions”; U.S. Patent Application No. 60/816,967, filed Jun. 28,2006, entitled “Zeaxanthin Production Methods and Novel CompositionsContaining Zeaxanthin”; U.S. Patent Application No. 60/872,072, filedNov. 30, 2006, entitled “Polysaccharide Compositions and Methods ofAdministering, Producing, and Formulating Polysaccharide Compositions”;and PCT Patent Application No: PCT/US2007/001319, filed Jan. 19, 2007,entitled “Nutraceutical Compositions from Microalgae and Related Methodsof Production and Administration”.

VII. Examples Example 1 Growth of Porphyridium cruentum and Porphyridiumsp.

Porphyridium sp. (strain UTEX 637) and Porphyridium cruentum (strainUTEX 161) were inoculated into autoclaved 2 liter Erlenmeyer flaskscontaining an artificial seawater media:

1495 ASW medium recipe from the American Type Culture Collection(components are per 1 liter of media)

NaCl 27.0 g MgSO₄•7H₂O 6.6 g MgCl₂•6H₂O 5.6 g CaCl₂•2H₂O 1.5 g KNO₃ 1.0g KH₂PO₄ 0.07 g NaHCO₃ 0.04 g 1.0M Tris-HCl buffer, pH 7.6 20.0 ml TraceMetal Solution (see below) 1.0 ml Chelated Iron Solution (see below) 1.0ml Distilled water bring to 1.0 L Trace Metal Solution: ZnCl₂ 4.0 mgH₃BO₃ 60.0 mg CoCl₂•6H₂O 1.5 mg CuCl2•2H₂O 4.0 mg MnCl₂•4H₂O 40.0 mg(NH₄)₆Mo₇O₂₄•4H₂O 37.0 mg Distilled water 100.0 ml Chelated IronSolution: FeCl₃•4H₂O 240.0 mg 0.05M EDTA, pH 7.6 100.0 ml

Media was autoclaved for at least 15 minutes at 121° C.

Inoculated cultures in 2 liter flasks were maintained at roomtemperature on stir plates. Stir bars were placed in the flasks beforeautoclaving. A mixture of 5% CO₂ and air was bubbled into the flasks.Gas was filter sterilized before entry. The flasks were under 24 hourillumination from above by standard fluorescent lights (approximately150 uE/m⁻¹/s⁻¹). Cells were grown for approximately 12 days, at whichpoint the cultures contained approximately of 4×10⁶ cells/mL.

Example 2

Dense Porphyridium sp. and Porphyridium cruentum cultures werecentrifuged at 4000 rcf. The supernatant was subjected to tangentialflow filtration in a Millipore Pellicon 2 device through a 1000 kDregenerated cellulose membrane (filter catalog number P2C01MC01).Approximately 4.1 liters of Porphyridium cruentum and 15 liters ofPorphyridium sp. supernatants were concentrated to a volume ofapproximately 200 ml in separate experiments. The concentratedexopolysaccharide solutions were then diafiltered with 10 liters of 1 mMTris (pH 7.5). The retentate was then flushed with 1 mM Tris (pH 7.5),and the total recovered polysaccharide was lyophilized to completion.Yield calculations were performed by the dimethylmethylene blue (DMMB)assay. The lyophilized polysaccharide was resuspended in deionized waterand protein was measured by the bicinchoninic acid (BCA) method. Totaldry product measured after lyophilization was 3.28 g for Porphyridiumsp. and 2.0 g for Porphyridium cruentum. Total protein calculated as apercentage of total dry product was 12.6% for Porphyridium sp. and 15.0%for Porphyridium cruentum.

Example 3 Increasing Solvent-Available Polysaccharide

A measured mass (approximately 125 grams) of freshly harvestedPorphyridium sp. cells, resuspended in a minimum amount of dH₂Osufficient to allow the cells to flow as a liquid, was placed in acontainer. The cells were subjected to increasing amounts of sonicationover time at a predetermined sonication level. Samples were drawn atpredetermined time intervals, suspended in measured volume of dH₂O anddiluted appropriately to allow visual observation under a microscope andmeasurement of polysaccharide concentration of the cell suspension usingthe DMMB assay. A plot was made of the total amount of time for whichthe biomass was sonicated and the polysaccharide concentration of thebiomass suspension. Two experiments were conducted with different timeintervals and total time the sample was subjected to sonication. Thefirst data set from sonication experiment 1 was obtained by subjectingthe sample to sonication for a total time period of 60 minutes in 5minute increments. The second data set from sonication experiment 2 wasobtained by subjecting the sample to sonication for a total time periodof 6 minutes in 1-minute increments. The data, observations andexperimental details are described below. Standard curves were generatedusing TFF-purified, lyophilized, weighed, resuspended Porphyridium sp.exopolysaccharide.

General Parameters of Sonication Experiments 1 and 2

Cells were collected and volume of the culture was measured. The biomasswas separated from the culture solution by centrifugation. Thecentrifuge used was a Form a Scientific Centra-GP8R refrigeratedcentrifuge. The parameters used for centrifugation were 4200 rpm, 8minutes, rotor #218. Following centrifugation, the biomass was washedwith dH₂O. The supernatant from the washings was discarded and thepelleted cell biomass was collected for the experiment.

A sample of 100 μL of the biomass suspension was collected at time point0 (OTP) and suspended in 900 μL dH₂O. The suspension was further dilutedten-fold and used for visual observation and DMMB assay. The time point0 sample represents the solvent-available polysaccharide concentrationin the cell suspension before the cells were subjected to sonication.This was the baseline polysaccharide value for the experiments.

The following sonication parameters were set: power level=8, 20 secondsON/20 seconds OFF (Misonix 3000 Sonicator with flat probe tip). Thecontainer with the biomass was placed in an ice bath to preventoverheating and the ice was replenished as necessary. The sample wasprepared as follows for visual observation and DMMB assay: 100 μL of thebiomass sample+900 μL dH₂O was labeled as dilution 1. 100 μL of (i)dilution 1+900 μL dH₂O for cell observation and DMMB assay.

Sonication Experiment 1

In the first experiment the sample was sonicated for a total time periodof 60 minutes, in 5-minute increments (20 seconds ON/20 seconds OFF).The data is presented in Tables 4, 5 and 6. The plots of the absorbanceresults are presented in FIG. 4.

TABLE 4 Sonication record for experiment 1. Time point Ser# (min)Observations (TP = time point) 1 0 Healthy red cells 2 5 Red colordisappeared, small greenish circular particles 3 10 Small particle,smaller than 5 minute TP 4 15 Small particle, smaller than 10 minute TP.Same observation as 10 minute time 5 20 Similar to 15 minute TP. Smallparticles; empty circular shells in the field of vision 6 25 Similar to20 minute TP 7 30 Similar to 25 minute TP, particles less numerous 8 35Similar to 30 minute TP 9 40 Similar to 35 minute TP 10 45 Similar to 40minute TP 11 50 Very few shells, mostly fine particles 12 55 Similar to50 minute TP. 13 60 Fine particles, hardly any shells

TABLE 5 Standard curve record for sonication experiment 1. Absorbance(AU) Concentration (μg) 0 Blank, 0 0.02 0.25 0.03 0.5 0.05 0.75 0.07 1.00.09 1.25

TABLE 6 Record of sample absorbance versus time points for sonicationexperiment 1. SAMPLE Solvent-Available TIME POINT Polysaccharide (MIN)(μg) 0 0.23 5 1.95 10 2.16 15 2.03 20 1.86 25 1.97 30 1.87 35 2.35 401.47 45 2.12 50 1.84 55 2.1 60 2.09

The plot of polysaccharide concentration versus sonication time pointsis displayed above and in FIG. 4. Solvent-available polysaccharideconcentration of the biomass (cell) suspension reaches a maximum valueafter 5 minutes of sonication. Additional sonication in 5-minuteincrements did not result in increased solvent-available polysaccharideconcentration.

Homogenization by sonication of the biomass resulted in an approximately10-fold increase in solvent-available polysaccharide concentration ofthe biomass suspension, indicating that homogenization significantlyenhances the amount of polysaccharide available to the solvent. Theseresults demonstrate that physically disrupted compositions ofPorphyridium for oral or other administration provide novel andunexpected levels or polysaccharide bioavailability compared tocompositions of intact cells. Visual observation of the samples alsoindicates rupture of the cell wall and thus release of insoluble cellwall-bound polysaccharides from the cells into the solution that ismeasured as the increased polysaccharide concentration in the biomasssuspension.

Sonication Experiment 2

In the second experiment the sample was sonicated for a total timeperiod of 6 minutes in 1-minute increments. The data is presented inTables 7, 8 and 9. The plots of the absorbance results are presented inFIG. 5.

TABLE 7 Sonication record for experiment 2. Time point Ser# (min)Observations 1 0 Healthy red-brown cells appear circular 2 1 Circularparticles scattered in the field of vision with few healthy cells. Redcolor has mostly disappeared from cell bodies. 3 2 Observation similarto time point 2 minute. 4 3 Very few healthy cells present. Red colorhas disappeared and the concentration of particles closer in size towhole cells has decreased dramatically. 5 4 Whole cells are completelyabsent. The particles are smaller and fewer in number. 6 5 Observationsimilar to time point 5 minute. 7 6 Whole cells are completely absent.Large particles are completely absent.

TABLE 8 Standard curve record for sonication experiment 2. Absorbance(AU) Concentration (μg) −0.001 Blank, 0 0.017 0.25 0.031 0.5 0.049 0.750.0645 1.0 0.079 1.25

TABLE 9 Record of sample absorbance versus time points for sonicationexperiment 2. SAMPLE Solvent-Available TIME POINT Polysaccharide (MIN)(μg) 0 0.063 1 0.6 2 1.04 3 1.41 4 1.59 5 1.74 6 1.78

The value of the solvent-available polysaccharide increases gradually upto the 5 minute time point as shown in Table 9 and FIG. 5.

Example 4 Alcohol Precipitation

Porphyridium sp. culture was centrifuged at 4000 rcf and supernatant wascollected. The supernatant was divided into six 30 ml aliquots. Threealiquots were autoclaved for 15 min at 121° C. After cooling to roomtemperature, one aliquot was mixed with methanol (58.3% vol/vol), onewas mixed with ethanol (47.5% vol/vol) and one was mixed withisopropanol (50% vol/vol). The same concentrations of these alcoholswere added to the three supernatant aliquots that were not autoclaved.Polysaccharide precipitates from all six samples were collectedimmediately by centrifugation at 4000 rcf at 20° C. for 10 min andpellets were washed in 20% of their respective alcohols. Pellets werethen dried by lyophilization and resuspended in 15 ml deionized water byplacement in a 60° C. water bath. Polysaccharide pellets fromnon-autoclaved samples were partially soluble or insoluble.Polysaccharide pellets from autoclaved ethanol and methanolprecipitation were partially soluble. The polysaccharide pellet obtainedfrom isopropanol precipitation of the autoclaved supernatant wascompletely soluble in water.

Example 5 Monosaccharide Analysis

Approximately 10 milligrams of purified polysaccharide from Porphyridiumsp. and Porphyridium cruentum (described in Example 3) were subjected tomonosaccharide analysis.

Monosaccharide analysis was performed by combined gaschromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl(TMS) derivatives of the monosaccharide methyl glycosides produced fromthe sample by acidic methanolysis.

Methyl glycosides prepared from 500 μg of the dry sample provided by theclient by methanolysis in 1 M HCl in methanol at 80° C. (18-22 hours),followed by re-N-acetylation with pyridine and acetic anhydride inmethanol (for detection of amino sugars). The samples were thenper-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80° C.(30 mins). These procedures were carried out as previously describeddescribed in Merkle and Poppe (1994) Methods Enzymol. 230: 1-15; York,et al. (1985) Methods Enzymol. 118: 3-40. GC/MS analysis of the TMSmethyl glycosides was performed on an HP 5890 GC interfaced to a 5970MSD, using a Supelco DB-1 fused silica capillary column (30 m 0.25 mmID).

Monosaccharide compositions were determined as follows:

TABLE 10 Porphyridium sp. monosaccharide analysis. Glycosyl residue Mass(μg) Mole % Arabinose (Ara) n.d. n.d. Rhamnose (Rha) 2.7 1.6 Fucose(Fuc) n.d. n.d. Xylose (Xyl) 70.2 44.2 Glucuronic acid (GlcA) n.d. n.d.Galacturonic acid (GalA) n.d. n.d. Mannose (Man) 3.5 1.8 Galactose (Gal)65.4 34.2 Glucose (Glc) 34.7 18.2 N-acetyl galactosamine (GalNAc) n.d.n.d. N-acetyl glucosamine (GlcNAc) trace trace Σ = 176.5

TABLE 11 Porphyridium cruentum monosaccharide analysis. Glycosyl residueMass (μg) Mole % Arabinose (Ara) n.d. n.d. Rhamnose (Rha) n.d. n.d.Fucose (Fuc) n.d. n.d. Xylose (Xyl) 148.8 53.2 Glucuronic Acid (GlcA)14.8 4.1 Mannose (Man) n.d. n.d. Galactose (Gal) 88.3 26.3 Glucose (Glc)55.0 16.4 N-acetyl glucosamine (GlcNAc) trace trace N-acetyl neuraminicacid (NANA) n.d. n.d. Σ = 292.1 Mole % values are expressed as molepercent of total carbohydrate in the sample. n.d. = none detected.

Example 6 Protein Measurement

Porphyridium sp. was grown as described. 2 liters of centrifugedPorphyridium sp. culture supernatant were autoclaved at 121° C. for 20minutes and then treated with 50% isopropanol to precipitatepolysaccharides. Prior to autoclaving the 2 liters of supernatantcontained 90.38 mg polysaccharide. The pellet was washed with 20%isopropanol and dried by lyophilization. The dried material wasresuspended in deionized water. The resuspended polysaccharide solutionwas dialyzed to completion against deionized water in a Spectra/Porcellulose ester dialysis membrane (25,000 MWCO). 4.24% of the solidcontent in the solution was proteins as measured by the BCA assay.

Example 7 Generation of Protein-Free Polysaccharide

Porphyridium sp. was grown as described. 1 liters of centrifugedPorphyridium sp. culture supernatant was autoclaved at 121° C. for 15minutes and then treated with 10% protease (Sigma catalog number P-5147;protease treatment amount relative to protein content of the supernatantas determined by BCA assay). The protease reaction proceeded for 4 daysat 37° C. The solution was then subjected to tangential flow filtrationin a Millipore Pellicon® cassette system using a 0.1 micrometerregenerated cellulose membrane. The retentate was diafiltered tocompletion with deionized water. No protein was detected in thediafiltered retentate by the BCA assay. See FIG. 6.

Optionally, the retentate can be autoclaved to achieve sterility if thefiltration system is not sterile. Optionally the sterile retentate canbe mixed with pharmaceutically acceptable carrier(s) and filled in vialsfor injection.

Optionally, the protein free polysaccharide can be fragmented by, forexample, sonication to reduce viscosity for parenteral injection as, forexample, an antiviral compound. Preferably the sterile polysaccharide isnot fragmented when prepared for injection as a joint lubricant.

Example 8 Heterotrophic Growth of Porphyridium

Cultures of Porphyridium sp. (UTEX 637) and Porphyridium cruentum(strain UTEX 161) were grown, to a density of 4×10⁶ cells/mL, asdescribed in Example 1. For each strain, about 2×10⁶ cells/mL cells perwell (˜500 uL) were transferred to 11 wells of a 24 well microtiterplate. These wells contained ATCC 1495 media supplemented with varyingconcentration of glycerol as follows: 0%, 0.1%, 0.25%, 0.5%, 0.75%, 1%,2%, 3%, 5%, 7% and 10%. Duplicate microtiter plates were shaken (a)under continuous illumination of approximately 2400 lux as measured by aVWR Traceable light meter (cat #21800-014), and (b) in the absence oflight. After 5 days, the effect of increasing concentrations of glycerolon the growth rate of these two species of Porphyridium in the light wasmonitored using a hemocytometer. The results are given in FIG. 2 andindicate that in light, 0.25 to 0.75 percent glycerol supports thehighest growth rate, with an apparent optimum concentration of 0.5%.

Cells in the dark were observed after about 3 weeks of growth. Theresults are given in FIG. 3 and indicate that in complete darkness, 5.0to 7.0% glycerol supports the highest growth rate, with an apparentoptimum concentration of 7.0%.

Example 9 Cosmeceutical Compositions

Porphyridium sp. (UTEX 637) was grown to a density of approximately4×10⁶ cells/mL, as described in Example 1. Approximately 50 grams of wetpelleted, and washed cells were completely homogenized usingapproximately 20 minutes of sonication as described. The homogenizedbiomass was mixed with carriers including, water, butylene glycol,mineral oil, petrolatum, glycerin, cetyl alcohol, propylene glycoldicaprylate/dicaprate, PEG-40 stearate, C11-13 isoparaffin, glycerylstearate, tri (PPG-3 myristyl ether) citrate, emulsifying wax,dimethicone, DMDM hydantoin, methylparaben, carbomer 940, ethylparaben,propylparaben, titanium dioxide, disodium EDTA, sodium hydroxide,butylparaben, and xanthan gum. The mixture was then further homogenizedto form a composition suitable for topical administration. Thecomposition was applied to human skin daily for a period of one week.

Example 10 Antibiotic Sensitivity

Approximately 4500 cells (300 ul of 1.5×10⁵ cells per ml) ofPorphyridium sp. and

Porphyridium cruentum cultures in liquid ATCC 1495 ASW media were platedonto ATCC 1495 ASW agar plates (1.5% agar). The plates contained varyingamounts of zeocin, sulfometuron, hygromycin and spectinomycin. Theplates were put under constant artificial fluorescent light ofapproximately 480 lux. After 14 days, plates were checked for growth.

Results were as follows:

Zeocin Conc. (ug/ml) Growth 0.0 ++++ 2.5 + 5.0 − 7.0 −

Hygromycin Conc. (ug/ml) Growth 0.0 ++++ 5.0 ++++ 10.0 ++++ 50.0 ++++

Specinomycin Conc. (ug/ml) Growth 0.0 ++++ 100.0 ++++ 250.0 ++++ 750.0++++

After the initial results above were obtained, a titration of zeocin wasperformed to more accurately determine growth levels of Porphyridium inthe presence of zeocin. Porphyridium sp. cells were plated as describedabove. Results are shown in FIG. 8.

Example 11 Nutritional Manipulation to Generate Novel Polysaccharides

Cells expressing an endogenous monosaccharide transporter, containing amonosaccharide transporter and capable of importing glucose, arecultured in ATCC 1495 media in the light in the presence of 1.0% glucosefor approximately 12 days. Exopolysaccharide is purified as described inExample 2. Monosaccharide analysis is performed as described in Example5.

Cells expressing an endogenous monosaccharide transporter, containing amonosaccharide transporter and capable of importing xylose, are culturedin ATCC 1495 media in the light in the presence of 1.0% xylose forapproximately 12 days. Exopolysaccharide is purified as described inExample 2. Monosaccharide analysis is performed as described in Example5.

Cells expressing an endogenous monosaccharide transporter, containing amonosaccharide transporter and capable of importing galactose, arecultured in ATCC 1495 media in the light in the presence of 1.0%galactose for approximately 12 days. Exopolysaccharide is purified asdescribed in Example 2. Monosaccharide analysis is performed asdescribed in Example 5.

Cells expressing an endogenous monosaccharide transporter, containing amonosaccharide transporter and capable of importing glucuronic acid, arecultured in ATCC 1495 media in the light in the presence of 1.0%glucuronic acid for approximately 12 days. Exopolysaccharide is purifiedas described in Example 2. Monosaccharide analysis is performed asdescribed in Example 5.

Cells expressing an endogenous monosaccharide transporter, containing amonosaccharide transporter and capable of importing glucose, arecultured in ATCC 1495 media in the dark in the presence of 1.0% glucosefor approximately 12 days. Exopolysaccharide is purified as described inExample 2. Monosaccharide analysis is performed as described in Example5.

Cells expressing an endogenous monosaccharide transporter, containing amonosaccharide transporter and capable of importing xylose, are culturedin ATCC 1495 media in the dark in the presence of 1.0% xylose forapproximately 12 days. Exopolysaccharide is purified as described inExample 2. Monosaccharide analysis is performed as described in Example5.

Cells expressing an endogenous monosaccharide transporter, containing amonosaccharide transporter and capable of importing galactose, arecultured in ATCC 1495 media in the dark in the presence of 1.0%galactose for approximately 12 days. Exopolysaccharide is purified asdescribed in Example 2. Monosaccharide analysis is performed asdescribed in Example 5.

Cells expressing an endogenous monosaccharide transporter, containing amonosaccharide transporter and capable of importing glucuronic acid, arecultured in ATCC 1495 media in the dark in the presence of 1.0%glucuronic acid for approximately 12 days. Exopolysaccharide is purifiedas described in Example 2. Monosaccharide analysis is performed asdescribed in Example 5.

Example 12 Increasing Solvent-Available Polysaccharide

128 mg of intact lyophilized Porphyridium sp. cells were ground with amortar/pestle. The sample placed in the mortar pestle was ground for 5minutes. 9.0 mg of the sample of the ground cells was placed in a microcentrifuge tube and suspended in 1000 μL of dH2O. The sample wasvortexed to suspend the cells.

A second sample of 9.0 mg of intact, lyophilized Porphyridium sp. cellswas placed in a micro centrifuge tube and suspended in 1000 μL of dH₂O.The sample was vortexed to suspend the cells.

The suspensions of both cells were diluted 1:10 and polysaccharideconcentration of the diluted samples was measured by DMMB assay. Upongrinding, the suspension of ground cells resulted in an approximately10-fold increase in the solvent-accessible polysaccharide as measured byDMMB assay over the same quantity of intact cells.

TABLE 12 Results of dimethylmethylene blue (DMMB) assay. Read 1 Read 2Avg. Abs Conc. Sample Description (AU) (AU) (AU) (μg/mL) Blank 0 −0.004−0.002 0  50 ng/μL Std., 10 μL; 0.5 μg 0.03 0.028 0.029 NA 100 ng/μLStd., 10 μL; 1.0 μg 0.056 0.055 0.0555 NA Whole cell suspension 0.0090.004 0.0065 0.0102 Ground cell suspension 0.091 0.072 0.0815 0.128

Reduction in the particle size of the lyophilized biomass byhomogenization in a mortar/pestle results in better suspension andincrease in the solvent-available polysaccharide concentration of thecell suspension.

Example 13 Decolorization of Biomass

A Porphyridium culture (UTEX 637) was grown as described in Example 1except that the culture media contained no Tris and 0.125 g/L potassiumnitrate, pH 7.6. The low nitrate culture was grown under approximately130 μE m⁻² s⁻¹ until the color changed from red to yellow-brown, whichtook approximately 3 weeks. After waiting for a further three days, theyellow-brown cells were harvested by centrifugation, hereinafterreferred to as “decolorized biomass” or “decolorized cell pellet”. Adeep red cell pellet generated from cells grown in normal ATCC 1495 ASWmedia was also generated as described in Example 1. The cell pelletswere washed with 0.5 L of distilled water, shell frozen in a dry iceacetone bath and lyophilized.

Determination of Color

The decolorized cell pellet had a yellow-brown appearance (as opposed tocells grown in full ATCC 1495 ASW media which have a deep redappearance). The lyophilized decolorized cells and lyophilized red cellsgrown in full ATCC 1495 ASW media were treated identically. 100 mg oflyophilized cell-pellets were resuspended in 4 ml of 1 M pH 7.6 Trisbuffer by vigorous vortexing. The suspensions were sonicated on iceusing a Misonix 3000 sonicator equipped with a micro-tip probe set at apower level of 6.5 for 90 seconds, pulsing for 30 seconds on 20 secondsoff (3 cycles). The cell debris was pelleted by centrifugation in amicrocentrifuge at 14,000 rpm for 5 minutes and the supernatantdecanted. This procedure was repeated twice more with 1M pH 7.6 Trisbuffer, and finally with 6M urea. No red color was observed in cellpellets or supernatant from the decolorized biomass after the secondextraction or from the cells grown in full ATCC 1495 ASW media after thefourth extraction. The respective supernatants were combined and broughtto a final volume of 75 ml with distilled water.

Absorbance spectra of the supernatants from the decolorized pellet andthe pellet from cells grown in full ATCC 1495 ASW media were recordedbetween 510 and 600 nM with a Pharmacia Ultraspec III spectrophotometerand a 1 cm path length cuvette.

The extinction coefficient for phycoerythrin is 5.26 ml.mg/cm at 545 nm(see for example Gantt and Lipschultz, Phycobilisomes of Porphyridiumcruentum; Biochemistry, 13, 2960, 1974). The concentration phycoerythrinwas calculated from the optical density at 545 nm (after subtracting thebackground due to scatter measured at 600 nm) as 46 mg/g dry-weight incells grown in ATCC 1495 ASW media and 4.7 mg/g in the decolorizedcells.

Example 14 Homogenization of Biomass

After Porphyridium biomass grown as described in Example 1 was recoveredby centrifugation and washed in deionizer water, nitrogen was bubbledthrough the paste for 30 minutes to displace dissolved oxygen andminimize subsequent oxidation. The paste was then passed through a model110Y Microfluidics Microfluidizer® at 22,000 PSI with cooling, and theprocess repeated until cell breakage was at least 50% as determined bymicroscopic examination. Nitrogen was once again bubbled through thepaste, which was then lyophilized after shell freezing in dry iceethanol. The dried cell mass was then ground to a fine powder with aBraun® kitchen homogenizer. This process can be performed withdecolorized biomass generated as described herein. Optionally,preservative(s) and/or carrier(s) suitable for topical administrationare added to the material, as well as fragrances and other materialsused in the art of skin care product formulation.

Example 15 Hyaluronidase Inhibition

Biotinylated hyaluronic acid (bHA) was covalently attached to the wellsof a 96-well plate. Samples containing hyaluronidase and various testmaterials were then added to the wells. The hyaluronidase degrades thebound hyaluronic acid, resulting in a decrease in the amount of biotincovalently linked to the well plate. At the end of the incubation periodthe reaction was stopped and the well plate was washed to remove thehyaluronidase. The remaining bHA was detected using an avidin boundperoxidase enzyme. When an appropriate substrate is added, theperoxidase enzyme generated a color signal in proportion to the amountof bHA. The color signal was measured spectrophotometrically, and wasinversely proportional to the amount of hyaluronidase activity in thesample. Thus, materials that inhibited hyaluronidase resulted in agreater color signal, since more of the bHA remained intact. Also seeFrost, G., I., Stern, R. A Microtiter-Based Assay for HyaluronidaseActivity Not Requiring Specialized Reagents. Analytical Biochemistry251, 263-269: 1997.

Preparation of Test Material Extracts

Test Material A was supplied as a powder type material. For this study,100 mg of this material was combined with either 5 ml of ethanol or 5 mlof ultrapure water in 15 ml centrifuge tubes. After combining, themixtures were vortexed, then placed onto a rocking platform forapproximately 30 minutes at room temperature, and then centrifuged at1,000 RPM for 5 minutes. The supernatants were then used at the finalconcentrations listed in the results section. Test Material B wassupplied as a thick, viscous solutions (3%).

Anti-Hyaluronidase Assay: Immobilization of bHA onto 96-Well Plates

A solution of sulfo-NHS (0.184 mg/ml) and bHA (0.2 mg/ml) was preparedin distilled water. 50 μl of this solution was then added to the wellsof a 96-well Covalink-NH plate. A solution of EDC (0.123 mg/ml) was thenprepared in distilled water and 50 μl of this solution was added to eachwell (which resulted in a final concentration of 10 μg/well bHA and 6.15μg/well of EDC). The well plate was then incubated overnight at 4±2° C.or for 2 hours at room temperature. After the incubation the plate waswashed three times with PBS containing 2 M NaCl and 50 mM MgSO₄.

Anti-Hyaluronidase Assay

Prior to the assay, the well plate was equilibrated with assay buffer(0.1 M formate [pH 4.5], 0.1 M NaCl, 1% Triton X-100, 5 mMsaccharolactone). The test materials were prepared in assay buffer at 2×their final concentration (heparin was used as a positive control, 1mg/ml final concentration). After removing the assay buffer from thewell plate used for equilibration, 50 μl of each of the prepared testmaterials was added to three wells on the well plate, followed by theaddition of 50 μl of assay buffer containing hyaluronidase will be addedto each well (0.1 mg/ml). Three additional wells were treated with 100μl of assay buffer alone (without test materials and withouthyaluronidase) and served as an index of zero hyaluronidase activity.After the addition of the test materials and enzyme, the plate wasincubated for 30 minutes at room temperature. At the end of theincubation period, 200 μl of 6 M guanidine-HCl was added to each well toterminate the reaction. The plate was then washed three times with PBScontaining 2 M NaCl, 50 mM MgSO₄ and 0.05% Tween 20.

During the 30 minute incubation, an avidin/biotin-peroxidase complex wasprepared in 10.5 ml of PBS containing 0.1% Tween 20 using an ABC kit.This mixture was incubated for at least 30 minutes prior to use. Afterthe plate was washed, 100 μl of the avidin/biotin-peroxidase solutionwas added to each well and the plate was incubated for another 30minutes at room temperature. The plate was washed three times with PBScontaining 2 M NaCl, 50 mM MgSO₄ and 0.05% Tween 20. After the finalwash, 100 μl of substrate solution (one 10 mg tablet of OPD in 10 ml of0.1 M citrate-PO₄ buffer supplemented with 7.5 μl of 30% H₂O₂) was addedto each well. The plate was incubated in the dark for 10-20 minutes andthen read at 460 nm using a plate reader. The substrate solution wasalso added to three wells that were not treated with test materials orthe avidin/biotin-peroxidase solution and were used as a blank for theabsorbance measurements.

TABLE 13 Results of anti-hyaluronidase assay. Treatment PercentInhibition  10% A Water Extract 86   5% MATERIAL A Water Extract 67   1%MATERIAL A Water Extract 29 1.5% MATERIAL B 93 0.5% MATERIAL B 81 0.1%MATERIAL B 70 0.1% Heparin 74 Negative Control 0 Test MaterialIdentification: MATERIAL A: Porphyridium sp. biomass homogenized (QuadroF10); cells grown as described in Example 1 Physical Description:Red/Purple powder Concentrations Tested: 10%, 5%, 1% (Extracted ineither ethanol or water) Test Material Identification: 3% MATERIAL B:Exopolysaccahride from Porphyridium sp. purified as described in Example2 Physical Description: Light tan, viscous liquid Concentrations Tested:1.5%, 1%, 0.5%, 0.1%

Example 16 Sulfated Derivative Polysaccharides

Porphyridium cruentum and Porphyridium sp. were grown in artificialseawater media essentially as described in Example 1 except that theamount of MgSO₄ was varied. Porphyridium sp.cells were grown in 17 mMMgSO₄ . Porphyridium cruentum was grown in 120 mM, 600 mM, 750 mM, 1M,and 2M MgSO₄. Cell division occurred at all concentrations.Polysaccharide was purified essentially as described in Example 2 fromthe 120 and 600 mM cultures, to the point where all soluble protein andsmall molecules were removed. Sulfur content was analyzed according toUS EPA SW846, Method 6010B, Inductively Coupled Plasma-Atomic EmissionSpectrometry. The polysaccharide purified from the 17, 120 and 600 mMcultures contained 3.57, 4.23 and 5.57% sulfur, respectively. It wasobserved that polysaccharides with higher percent sulfate by weightexhibited stronger gelling properties than polysaccharides with a lowerpercent sulfate by weight when the two preparations were generated atthe same polysaccharide concentration. For example, at a 1%concentration the polysaccharide containing 5.57% sulfur held its shapeand moved as a gelatinous unit whereas the polysaccharide with a 3.57percent sulfur by weight at 1% flowed as a viscous liquid. The increasedgelling properties provide added benefits for skin care compositions asthey can form gels in products at lower concentrations.

Example 17 Monosaccharide Analysis

Porphyridium cruentum was cultured as described in Example 1 except that(a) the amount of KNO₃ per liter of media was approximately 150 g/L; (b)the media contained no Tris; and (c) the media contained approximately0.14 g/L KH₂PO₄. The cells lost all detectable red coloration afterapproximately three weeks of growth, and turned to a yellow shade.Exopolysaccharide was purified essentially as described in Example 2.Monosaccharide analysis was performed essentially as described inExample 5.

Monosaccharide composition of the exopolysaccharide was as follows:

Glycosyl residue Mass (μg) Mole % Arabinose (Ara) n.d. n.d. Rhamnose(Rha) n.d. n.d. Fucose (Fuc) n.d. n.d. Xylose (Xyl) 137.8 41.5Glucuronic Acid (GlcA) 18.7 4.3 Mannose (Man) trace trace Galactose(Gal) 133.2 33.4 Glucose (Glc) 83.2 20.8 Unknown Sugar n.d. n.d.N-acetyl glucosamine (GlcNAc) n.d. n.d. N-acetyl neuraminic acid (NANA)n.d. n.d. Σ = 372.9

The exopolysaccharide contained significantly different and moreadvantageous monosaccharide composition from those grown under standardconditions shown in Example 5. The composition is a preferredcomposition for skin care products. For example, the ratio of glucose toxylose is higher in the polysaccharide from bleached cells.

Example 18 Polysaccharide Bead Production

Two solutions of polysaccharide from Porphyridium cruentum (0.5% w/v) inwater, prepared as described in Example 2 except flushed with distilledwater rather than 1 mM Tris, were dried under air flow at 60° C. untilconverted to a solid translucent film. One sample was isolated fromPorphyridium cruentum grown in ATCC 1495 media, while the other was fromPorphyridium cruentum grown in ATCC 1495 media with the exception thatthe KNO₃ was approximately 0.15 g/L (labeled as “1495 low N”). Theresulting films were then heated under vacuum (≧25 in Hg) atapproximately 150° C. overnight to form dried polysaccharide. The driedpolysaccharide was then ground in a pestle and mortar. A thirdpolysaccharide sample (0.5% w/v) in water, isolated from Porphyridiumcruentum grown in ATCC 1495 media, prepared as described in Example 2except flushed with distilled water rather than 1 mM Tris, waslyophilized and not ground.

100 mg of dried, ground polysaccharide from each sample was resuspendedin 2.5 ml of water. 100 mg of lyophilized polysaccharide was alsoresuspended in 2.5 ml of water. The suspensions were centrifuged at4,400 rpm in a Form a Scientific Centra-GP8R refrigerated centrifuge.The parameters used for centrifugation were 4200 rpm, rotor #218, 20minute spin. As shown in FIG. 16( c), there was a swollen, insoluble gellayer and a clear supernatant in the samples that were dried at 150° C.but not in the samples that were lyophilized or dried at 105° C. Thepolysaccharide dried at 150° C. did not go into solution, but ratherstayed insoluble despite significant swelling in size.

The percentage insoluble polysaccharide in the 150° C. dried samples wasmeasured by separating the insoluble and supernatant fractions,lyophilizing the separated fractions, and weighing the dried residualpolysaccharide from each fraction. The percentage insolublepolysaccharide was then calculated as a percentage of the totalpolysaccharide from both fractions. While the polysaccharide from thesamples that were originally dried by lyophilization and drying at 105°C. were 100% soluble, the low N and ATCC 1495 polysaccharide samplesdried at 150° C. were 75% and 86% insoluble, respectively. Independentexperiments demonstrated that material dried at 125° C. was completelysoluble.

Example 19 Polysaccharide Bead Properties

Insoluble polysaccharide preparations, prepared essentially as describedin Example 18, were tested for (a) the ability to swell in size overtime and (b) the ability to bind soluble polysaccharide and remove itfrom solution.

Samples were resuspended in distilled water as described in Example 18.Samples were centrifuged as described in Example 18 at various timepoints. The volume of the insoluble gel layer was measured, followed byresuspension of the material and incubation at room temperature untilthe next centrifugation point. Results are shown in FIG. 17. The resultsdemonstrate that the dried polysaccharide beads continue to swell involume for at least 4 hours and as long as 18 hours after initialexposure to water.

The concentration of polysaccharide in solution in the supernatant wasmeasured at each time point using the DMMB assay as described in Example3. Results are shown in FIG. 17( b). The results demonstrate that thepolysaccharide beads bind soluble polysaccharide and remove it fromsolution for at least 4 hours after initial exposure to water. Theswelling and binding of soluble polysaccharide is a useful property fortopical application to human skin and is stimulated by transepidermalwater loss.

Example 20 Transformation of Porphyridium and Genotyping

The Porphyridum glycoprotein promoter, SEQ ID NO:21, was cloned inoperable linkage with a zeocin resistance ble cDNA with small amounts offlanking sequence (SEQ ID NO:37), with the far, 5′ region of theglycoprotein promoter directly adjacent to the pBlusecript SacI site andthe far 3′ region of the CMV 3′UTR (SEQ ID NO:32) adjacent to thepBlusecript KpnI site. The CMV 3′UTR was also in operable linkage withthe ble cDNA. The plasmid was linearized by KpnI, which does cut in thepromoter, ble cDNA, or 3′UTR, prior to transformation.

The biolistic particle delivery system PDS 1000/He (Bio-Rad, USA) wasused for transformation. Porphyridium sp. culture was grown tologarithmic phase (˜2×10⁶ cells/mL) in liquid ATCC 1495 media undercontinuous light (approximately 75 umol/photons/m²). Cells from thisculture were harvested at 4,000 rpm at room temperature. The cell pelletwas washed twice with sterile distilled water. Cells were resuspended infresh ATCC 1495 media to the same cell density i.e. ˜2×10⁶ cells/mL andincubated in the dark for 16 hours. The dark adapted cells were thenharvested at 4000 rpm at room temperature, resuspended in fresh ATCC1495 media to a density of ˜2×10⁸ cells/mL. Approximately 1×10⁸ cellswere transferred to each ATCC 1495 agarose plate. Filter sterilized DNAfrom the plasmids was coated onto 550 nm gold particles (catalog numberS04e, Seashell Technology, USA) according to the manufacturer'sprotocol. For each of the particle bombardments, 1 ug of plasmid DNA wasused. The negative controls were bombarded in identical fashion withgold particles coated with a plasmid containing the Porphyridumglycoprotein promoter, SEQ ID NO:21, and the CMV 3′UTR, (SEQ ID NO:32),with no zeocin resistance gene. Each of the particle bombardments wereperformed using 1350 psi rupture disks, at bombardment distance of 9 cm,and under 28 in. Hg vacuum. The bombarded cells were scraped off theplates, and transferred to 100 ml of fresh ATCC 1495 media, and shakenunder continuous light (approximately 75 umol/m²) for 3 days. Followingrecovery, the cells were harvested at 4,000 rpm at room temperature, andplated onto ATCC 1495 plates supplemented with 30 ug/mL Zeocin(Invitrogen, Carlsbad, Calif., USA) at a cell density of 1×10⁷cells/plate. These plates were incubated under light (approximately 25umol/m²) for 4-5 weeks. Zeocin resistant colonies growing on theseplates were scored as transformants and transferred onto fresh ATCC 1495plates supplemented with 30 ug/mL Zeocin (Invitrogen, Carlsbad, Calif.,USA) for growth and analysis.

Zeocin resistant colonies appeared after 2-3 weeks. Genotyping withprimers specific to the zeocin resistance gene was performed on genomicDNA isolated from zeocin resistant colonies. Results from genotyping ofone strain (referred to herein and labeled as “transformant #2” in FIG.14) indicated that the zeocin resistance gene was present. A band of thecorrect size was amplified. Results are shown in FIG. 14 and discussedin more detail in Example 20.

Example 21 Transformation of Porphyridium, Genotyping, and Southern BlotAnalysis

The zeocin resistance plasmid described in Example 20 and a secondplasmid that was identical with the exception that it contained a cDNAfor a human GLUT1 glucose transporter (SEQ ID NO:25) instead of the blecDNA were combined in a co-transformation experiment carried outessentially as described in Example 20 except that both the zeocinresistance and GLUT1 plasmids were both adhered to the gold beads. Azeocin resistant colony (referred to herein as transformant #1) wasselected for further analysis. Genomic DNA was extracted from wild typePorphyridium sp. and transformant #1.

Genotyping was performed on genomic DNA extracted from wild type,transformant #1, and transformant #2 DNA with plasmid DNA used as atemplate positive control and water in place of DNA as a templatenegative control. A segment of the Porphyridium glycoprotein (GLP) genepromoter was used as a target positive control. The following primersets were used for the genotyping PCR: Ble-FWD (SEQ ID NO:26) andBle-REV (SEQ ID NO:27), GLP-FWD (SEQ ID NO:28) and (SEQ ID NO:29),GLUT1-FWD (SEQ ID NO:30) and GLUT1-REV (SEQ ID NO:31). The PCR profileused was as follows: 94° C. denaturation for 5 min; 35 cycles of 94° C.for 30 sec, 51° C. or 60° C. (51° C. for glycoprotein gene & GLUT1 and60° C. for ble) for 30 sec, 72° C. for 2 min; 72° C. for 5 min. Resultsare shown in FIG. 14. FIG. 14( a) demonstrates that the ble gene waspresent in both transformants, as the expected 300 bp product wasgenerated. FIG. 14( b) demonstrates that the genomic DNA extraction andamplification was working, as the expected 948 bp glycoprotein promoterfragment was generated. FIG. 14( c) demonstrates that the GLUT1 gene waspresent transformant #1, as the expected 325 bp product was generated.DNA ladder was from BioNexus, Inc., All Purpose Hi-Lo DNA Marker,Catalog No: BN2050.

Specific bands can be amplified from residual plasmid DNA adhered to theoutside of cells on transformation plates. Additionally, plasmids thathave not been linearized can be maintained as episomes for a period oftime before being degraded and can serve as template during PCR despitenot having been integrated into a chromosome of a host organism. In bothcases, microalgal strains may genotype positive despite the absence ofstable chromosomal integration of the vector. Antibiotic resistantstrains are known to arise due to mutagenesis caused by chromosomaldamage from biolistic particles, electroporation conditions, and randomgenetic variation that is known to occur in microbial organisms.Southern blot analysis was performed to conclusively confirm theintegration of the GLUT1 construct into the genome of transformant #1.

Southern blot analysis was performed on transformant #1. 20 μg genomicDNA from wild type and transformant#1 were individually digested withHinc II, Sac I, Xho I and separated on a 1% agarose gel. DNA wastransferred onto Nylon membrane (Hybond N+, Amersham Biosciences). A1495 bp fragment containing the entire coding region of the GLUT1 genewas used as a probe. DIG labeled probes were generated for each probefragment using the DIG High Prime DNA labeling and detection Kitaccording to the manufacturers instructions (Roche). The hybridizingbands were detected using the calorimetric detection reagents providedby the manufacturer. FIG. 15 demonstrates that the GLUT1 plasmid wasstably integrated into the genome of transformant #1 while no detectablesignal arose from wild type genomic DNA. As would be expected for aplasmid integrating into a chromosome of an organism, the specific bandwas in a different position for each different restriction enzyme usedto digest the genomic DNA. This data conclusively demonstrates a speciesof the genus Porphyridium containing an exogenous gene encoding acarbohydrate transporter integrated into a chromosome of the organism.In this embodiment the carbohydrate transporter gene is in operablelinkage with a promoter endogenous to a species of the genusPorphyridium. In some other embodiments embodiment the carbohydratetransporter gene is in operable linkage with a promoter endogenous to aRhodophyte.

Example 22 Production of Materials from Microalgae

Materials from Porphyridium were generated to assess their utility ascomponents of skin care compositions. Materials BM, LS PS, and HS PSwere generated and tested as referenced in this and following Examples.BM is Porphyridium sp. biomass, grown essentially as described inExample 1, which was washed once with ditilled water, run twice througha microfluidizer (Microfluidics Inc, Newton, Mass., U.S.A.), andlyophilized as follows: using a Microfluidics Microfluidizer® (model#110Y) pressurized with nitrogen, washed biomass material was pumpedthrough an 87 um orifice at 22,000 psi twice. The product was kept onice at all times. Nitrogen gas was bubbled through the final productwhile mixing for 10 min. Snap freeze for storage or shell freeze andlyophilize. The BM material was then treated as described in eachexample.

LS PS was polysaccharide from Porphyridium sp. HS PS was polysaccharidefrom Porphyridium cruentum. Both were purified essentially as describedin Example 2. The cells grown for preparation of LS PS and HS PS weregrown essentially as described in Example 1 except that the sulfate inthe media was 17 mM for the LS PS and 600 mM for the HS PS (alsodescribed in Example 16).

Example 23 UVB Protective Properties of Materials from Microalgae

TT dimer-UV exposure assay: The testing system used for this assay wasthe MatTek EpiDerm®, a skin model that consists of normal human-derivedepidermal keratinocytes cultured to form a multilayered, highlydifferentiated model of the human epidermis. For this study, the tissueswere treated topically overnight with either test materials, 1 mM Trolox(positive control), or left untreated (negative control). On thefollowing day, the tissues were exposed to UVB (300 mJ/cm²). Followingthe exposures the DNA was extracted from the EpiDerm tissues and assayedfor thymine dimer content. Samples of the DNA were immobilized on asolid membrane support and incubated with an antibody that recognizesthymidine dimers in double stranded DNA. The primary antibody wasdetected using a secondary antibody conjugated to an alkalinephosphatase enzyme followed by the addition of a substrate that thealkaline phosphatase enzyme uses to generate a chemiluminescent signal.The light generated by this reaction was captured using film with theintensity of the light signal being proportional to the amount of thethymine dimers present in the sample.

Test material BM was Porphyridium sp. biomass that had been homogenizedwith a Microfluidizer® twice and then lyophilized. For this study, 100mg of this material was combined with 5 ml of ultrapure water in 15 mlcentrifuge tubes. After combining, the mixture was vortexed, then placedonto a rocking platform for approximately 30 minutes at roomtemperature, and then centrifuged at 1,000 RPM for 5 minutes. Thesupernatant was then used at a final concentration of 10% and 5%. Thetwo remaining test materials, LS PS and HS PS were supplied as thick,viscous solutions (3 g/100 mL). LS PS53 was tested at the finalconcentrations of 1.5%, 0.5%, and 0.1%, while PS133 was tested at thefinal concentration of 0.1%.

Prior to use, the MatTek EpiDerm® tissues were removed from theagarose-shipping tray and placed into a 6-well plate containing 0.9 mlof culture medium (MatTek EPI-100 culture media) according to themanufacturer. The tissues were allowed to incubate for at least 1 hourat 37±2° C. and 5±1% CO₂. After this initial incubation, the culturemedia was replaced with fresh, pre-warmed EPI-100 media and 100 μl oftest material, 1 mM Trolox or PBS (negative control) was applied. Thetissues were then incubated overnight at 37±2° C. and 5±1% CO₂. On thefollowing day, the tissues were exposed to 300 mJ/cm² of UVB energy at300 nm. After the UVB exposure the tissues were collected and DNA wasimmediately extracted.

DNA extraction was performed using the Qiagen, DNEasy Kit. Singletissues were placed into 1.5 ml centrifuge tubes containing 180 μl ofLysis Buffer One. After mincing the tissues with a pair of fine tippedscissors, 20 μl of Proteinase K was added to the tube and the tube wasincubated overnight at 55±2° C. with occasional mixing/vortexing. Afterthe Proteinase K digestion, 200 μl of Lysis Buffer Two was added to thetube and the tube was incubated at 70±2° C. for approximately 10minutes. Next, the DNA was precipitated by the addition of 200 μl of100% ethanol. The precipitated DNA was washed to remove cellular debrisby applying the mixture to a DNEasy Spin Column and centrifuging thesample in a 2 ml collection tube at 8,000 RPM for 1 minute. The flowthrough and collection tube was discarded, and 500 μl of Wash Buffer Onewas added to the spin column and the column was placed into a newcollection tube and centrifuged at 8,000 RPM for 1 minute. The flowthrough and collection tube was again discarded, and 500 μl of WashBuffer Two was added to the spin column and the column was placed into anew collection tube and centrifuged at 14,000 RPM for 3 minutes. Thespin column was then placed into a new 1.5 ml centrifuge tube and 110 μlof Elution Buffer was added to the column. The column was incubated for1 minute at room temperature and then centrifuged at 8,000 RPM for 1minute.

Extracted DNA was quantified via a fluorometric assay. A 10 μl aliquotof the DNA sample was mixed with 1.0 ml of Assay Buffer (2 M NaCl, 50 mMsodium phosphate, pH 7.4) and 100 μl of this diluted sample wastransferred to a well in a black 96-well plate. A series of DNAstandards (0, 100, 200, 300, 400 and 500 ng) was also transferred towells in a 96-well plate (in duplicate). Finally, 100 μl of diluteHoechst 33258 dye (0.006 mg/ml in Assay Buffer) was added to each welland the fluorescence intensity of each well was determined using anexcitation wavelength of 355 nm and an emission wavelength of 485 nm.

Aliquots of DNA (400 ng in 2×SSC [20× stock SSC: 3 M NaCl, 0.3 M sodiumcitrate, pH 7.0]) was loaded onto a membrane via microfiltrationblotting. After loading, the membrane was washed once in 2×SSC and thenbaked for 30 minutes at 80° C. to crosslink the DNA to the membrane. Themembrane was then incubated for 1 hour in blocking solution (TBS [20 mMTris, pH 7.5, 500 mM NaCl] supplemented with 5% non-fat milk protein,0.2% polyvinylpyrolidone, and 0.2% ficol), and then briefly washed twicein TBS-T (TBS with 0.1% non-fat milk protein and 0.1% Tween 20). Themembrane was then incubated overnight (4° C.) with an antibody thatrecognizes thymine dimers diluted in TBS-T. On the following day, themembrane was washed 3 times with TBS-T (20 minutes per wash) and thenincubated with an AP-conjugated secondary antibody for 1-2 hours at roomtemperature. After this incubation period the membrane was washed asdescribed above.

Near the end of the final wash the chemiluminescence reagent wasprepared. At the end of the last wash, all of the TBS-T was drained fromthe membrane and the chemiluminescent substrate was applied to themembrane and it was allowed to sit for approximately 1 minute. Themembrane was then wrapped in Saran foil and taped inside of a filmcassette. In a dark room a piece of film was inserted into the cassetteand exposed for various amounts of time, starting with 10 seconds andincreasing or decreasing in appropriate increments, until obtaining thenecessary exposures. After exposure, the films were analyzed viadensitometry. To quantify the amount of DNA present, a standard curvewas generated using known concentrations of DNA and their respectivefluorescence intensity (measured in RFUs or relative fluorescenceunits). A regression analysis was performed to establish the line thatbest fits these data points. The RFUs for each unknown sample were usedto estimate the amount DNA. Mean densitometric values, expressed inoptical density units, were determined for each treatment.

The results for the thymidine dimer assay are presented in Table 14 andFIG. 11( a). The values are expressed as optical density units. Thevalues are presented as mean values±standard deviation.

TABLE 14 Thymidine dimer assay. Treatment Optical Density  10% BM  85 ±11   5% BM  140 ± 15 1.5% LS PS 140 ± 3 0.5% LS PS  152 ± 16 0.1% LS PS 150 ± 13 0.1% HS PS 160 ± 2   1 mM Trolox 107 ± 1 Untreated 196 ± 6Non-UVB Exposed  39 ± 4

The results of this study indicate that all three of the test materialssignificantly reduced the amount of TT dimer formation.

Example 24 Procollagen Synthesis Stimulating Properties of Materialsfrom Microalgae

A fibroblast cell culture model was used to assess the ability of thepolysaccharide from Porphyridium to exert an effect on procollagensynthesis. Fibroblasts are the main source of the extracelluar matrixpeptides, including the structural protein collagen. Procollagen is alarge peptide synthesized by fibroblasts in the dermal layer of the skinand is the precursor for collagen. As the peptide is processed to form amature collagen protein, the propeptide portion is cleaved off (type IC-peptide). Both the mature collagen protein and the type I C-peptidefragment are then released into the extracellular environment. Ascollagen is synthesized the type I C-peptide fragment accumulates intothe tissue culture medium. Since there is a 1:1 stoichiometric ratiobetween the two parts of the procollagen peptide, assaying for type IC-peptide reflects the amount of collagen synthesized. Type 1 C-peptidewas assayed via an ELISA based method. Test material LS PS was suppliedas a thick, viscous solution (3 g/100 mL). This material was used at a0.1% final concentration. The anti-collagen antibody used wasProcollagen Type I: N-18 antibody, catalog number sc-8785, Santa CruzBiotechnology. The secondary antibody used was anti-goat conjugated withalkaline phosphatase catalog number sc-2355, Santa Cruz Biotechnology.

Fibroblasts were seeded into the individual wells of a 12 well plate in1.0 ml of Fibroblast Growth Media (FGM: DMEM supplemented with 2% FBS, 5ng/ml human recombinant growth factor, 5 ug/ml insulin, 50 ug/mlgentamicin, and 0.5 ug/ml Amphotericin-B) and incubated overnight at37±2° C. and 5±1% CO₂. On the following day the media was removed viaaspiration to eliminate any non-adherent cells and replaced with 1.0 mlof fresh FGM. The cells were grown until confluent with a media changeevery 48 to 72 hours. Upon reaching confluency the cells were treatedfor 24 hours with DMEM supplemented with 1.5% FBS to wash out anyeffects from the growth factors included in the normal culture media.After this 24-hour wash out period the cells were treated with 0.1% LSPS dissolved in FGM with 1.5% FBS. 4 mM sodium butyrate was used as apositive control. Untreated cells (negative controls) received FGM with1.5% FBS. The cells were incubated for 48 hours and at the end of theincubation period cell culture medium was collected and either storedfrozen (−75° C.) or assayed immediately. Materials were tested intriplicate.

A series of type I C-peptide standards was prepared ranging from 0 ng/mlto 640 ng/ml. Next, an ELISA microplate was prepared by removing anyunneeded strips from the plate frame followed by the addition of 100 μlof peroxidase-labeled anti procollagen type I-C peptide antibody to eachwell used in the assay. Twenty (20) μl of either sample (collectedtissue culture media) or standard was then added to appropriate wellsand the microplate was covered and allowed to incubate for 3±0.25 hoursat 37° C. After the incubation the wells were aspirated and washed threetimes with 400 μl of wash buffer. After the last wash was removed 100 μlof peroxidase substrate solution (hydrogen peroxide+tetramethylbenzidineas a chromagen) was added to each well and the plate was incubated for15±5 minutes at room temperature. After the incubation 100 μl of stopsolution (1 N sulfuric acid) was added to each well and the plate wasread using a microplate reader at 450 nm.

To quantify the amount of procollagen present, a standard curve wasgenerated using known concentrations of procollagen. A regressionanalysis was be performed to establish the line that best fit the datapoints. Absorbance values for the test and positive control samples wasused to determine the amount of procollagen present. The values arepresented in mean ng/ml±the standard deviation of the mean. 0.1% LS PSincreased procollagen synthesis compared to untreated cells, as seenbelow in Table 15 and in FIG. 11( b).

TABLE 15 Procollagen assay. Treatment Type I C-Peptide ng/ml 0.1% LS PS 1448 ± 113 4 mM Na Butyrate 1425 ± 81 Untreated 1151 ± 96

Example 25 Elastin Synthesis Stimulating Properties of Materials fromMicroalgae

A fibroblast cell culture model was used to assess elastin synthesis.Elastin is the main component of a network of elastic fibers that givetissues their ability to recoil after a transient stretch. This proteinis released by fibroblasts (soluble elastin) into the extracellularspace where it is then cross-linked to other elastin proteins to form anextensive network of fibers and sheets (insoluble elastin). Solubleelastin can be readily measured from cell culture medium via acompetitive ELISA based method.

Test material BM was supplied as a powder type material. 100 mg of BMwas combined with 5 ml of ultrapure water in 15 ml centrifuge tubes.After combining, the mixtures were vortexed, then placed onto a rockingplatform for approximately 30 minutes at room temperature, and thencentrifuged at 1,000 RPM for 5 minutes. The supernatant for each mixturewas then used at final concentrations indicated in FIG. 12( a). LS PSand HS PS was supplied as a thick, viscous solution (3 g/100 mL). Thismaterial was used at final concentrations indicated in FIG. 12( a).

Fibroblasts were seeded into the individual wells of a 12 well plate in1.0 ml of Fibroblast Growth Media (FGM) and incubated overnight at 37±2°C. and 5±1% CO₂. On the following day the media was removed viaaspiration to eliminate any non-adherent cells and replaced with 1.0 mlof fresh FGM. The cells were grown until confluent, with a media changeevery 48 to 72 hours. Upon reaching confluency the cells were treatedfor 24 hours with DMEM supplemented with 1.5% FBS to wash out anyeffects from the growth factors included in the normal culture media.After this 24-hour wash out period the cells were treated with the testmaterials at the specified concentrations dissolved in FGM with 1.5%FBS. 4 mM sodium butyrate was used as a positive control for elastinsynthesis. Untreated cells (negative controls) received FGM with 1.5%FBS. The cells were incubated for 48 hours and at the end of theincubation period cell culture medium was collected and either storedfrozen (−75° C.) or assayed immediately. Materials were tested intriplicate.

Soluble α-elastin was dissolved in 0.1 M sodium carbonate (pH 9.0) at aconcentration of 1.25 μg/ml. 150 μl of this solution was then applied tothe wells of a 96-well maxisorp Nunc plate and the plate was incubatedovernight at 4° C. On the following day the wells were saturated withPBS containing 0.25% BSA and 0.05% Tween 20. The plate was thenincubated with this blocking solution for 1 hour at 37° C. and thenwashed two times with PBS containing 0.05% Tween 20.

A set of α-elastin standards was generated ranging from 0 to 100 ng/ml.180 μl of either standard or sample was then transferred to a 650 μlmicrocentrifuge tube. The anti-elastin antibody was the C-21 antibody,catalog number sc-17581, from Santa Cruz Biotechnology. The secondaryantibody used was anti-goat conjugated with alkaline phosphatase catalognumber sc-2355, Santa Cruz Biotechnology. An anti-elastin antibodysolution was prepared (the antibody was diluted 1:100 in PBS containing0.25% BSA and 0.05% Tween 20) and 20 μl of the solution was added to thetube. The tubes were then incubated overnight at 4±2° C. On thefollowing day, 150 μl was transferred from each tube to the 96-wellelastin ELISA plate, and the plate was incubated for 1 hour at roomtemperature. The plate was then washed 3 times with PBS containing 0.05%Tween 20. After washing, 200 μl of a solution containing a peroxidaselinked secondary antibody diluted in PBS containing 0.25% BSA and 0.05%Tween 20 was added, and the plate was incubated for 1 hour at roomtemperature. After washing the plate three times as described above, 200μl of a substrate solution was added and the plate was incubated for 10to 30 minutes in the dark at room temperature. After this finalincubation the plate was read at 460 nm using a plate reader.

To quantify the amount of elastin present, a standard curve wasgenerated using known concentrations of elastin. A regression analysiswas performed to establish the line that best fit these data points.Absorbance values for the test and control samples was used to determinethe amount of elastin present in each sample. Both the 0.5% and 0.1%concentrations of LS PS and HS PS induced an increase in elastinsynthesis.

Example 26 Antiinflammatory Properties of Materials from Microalgae

Microalgal polysaccharide materials were tested for their ability toinfluence the migration of polymorphonucelar (PMN) leukocytes inresponse to chemotractant substances. Leukocyte migration is essentialfor leukocyte accumulation at sites of inflammation. During theinflammatory response, leukocytes migrate towards the source of achemotractant substance in a process called chemotaxis. In vitro methodsto study chemotaxis often use a membrane based approach, where achemoattractant is placed on one side of a membrane and PMN leukocytesare placed on the other. Chemotaxis is then measured by quantifying thenumber of leukocytes which then migrate through the filter towards thechemotractant substance.

For this study, human PMN leukocytes were isolated via densitycentrifugation from freshly drawn blood. The PMN cells were loaded witha Calcein AM, a fluorescent dye. While the PMN cells were being labeled,the bottom chamber of a chemotaxis chamber was filled with PBScontaining FMLP, a chemotractant substance. A membrane filter was placedover the bottom wells, after which a small aliquot of fluorescentlylabeled PMN cells was placed on top, along with the LS PS and HS PS testmaterials and positive control materials. The chemotaxis chamber was beincubated and at the end of the incubation period the fluorescence ofthe bottom wells was read and used as an index of PMN migration. Testmaterial LS PS and HS PS, were supplied as thick, viscous solutions (3g/100 mL). These materials were used at a final concentration of 1.5%,0.5% and 0.1%.

Heparinized whole blood (20-30 ml) was collected from a healthy humandonor, layered over a density gradient (Histopaque 1077 and Histopaque1119) and centrifuged at 400 g for 30 min. The PMN rich fraction wasremoved, washed twice in with phosphate buffered saline (PBS) and thenresuspended in 5.0 ml RPMI-1640 without phenol red supplemented with 10%heat-treated fetal calf serum (RPMI-FCS). Calcein AM (5 μg/ml final) wasadded to the cell suspension and then the cells were incubated for 30min at 37° C. After the incubation, the PMN cells were washed twice withPBS, counted and resuspended in RPMI-FCS at 1−5×10⁶ cells/ml.

A disposable 96-well chemotaxis chamber was used to measure PMNmigration (chemotaxis chambers manufactured by Neuroprobe Inc.,Gaithersburg, Md., catalog number 101-5). To set up the 96-well chamber,the wells in the microplate (bottom chamber) were filled with 29 μl ofFMLP (0.1 μM) diluted in PBS with 0.1% serum albumin. Negative controlwells were prepared with PBS and albumin (no FMLP added). Amphotericin B(2 μg/ml) was used as a positive control (inhibits PMN migration). Inaddition, 3 wells were filled with 12.5 μl of fluorescently labeled PMNcells and 12.5 μl of PBS with 0.1% albumin. These latter wells were usedas an index to represent 100% migration by the PMN cells.

Test materials were prepared in RPMI-FCS at 2× their desired finalconcentration. RPMI-FCS without test materials was used as anothernegative control. Aliquots of the test materials were combined with anequal volume of the fluorescently labeled PMN cells. A chemotaxismembrane was then placed over the bottom wells of the chemotaxis chamberand anchored into place. 25 μl of the PMN/test material combination wasthen spotted onto the membrane above each of the bottom wells (materialswere tested in triplicate) and the well plate was incubated for 1 hourat 37±2° C. and 5% CO₂. At the end of the incubation, the top of thechemotaxis membrane was wiped clean with a tissue to remove anynon-migrating cells and the bottom wells of the chemotaxis chamber wereread using a fluorescent plate reader (485 nm excitation, 530 nmemission).

The mean fluorescence of the three wells filled directly withfluorescent PMN cells was determined and used to represent 100%migration. The mean fluorescence of the test material treated PMN cellswere then determined (Test Material Migration). The fluorescent valueswere corrected for non-chemotaxis migration by subtracting the meanfluorescent measurements for the wells where FMLP was not present as achemotractant. Percent PMN migration was then calculated using thefollowing equation: ((Test Material Migration (mean RFU))/(100%Migration (mean RFU)))×100

The results for the PMN migration assay are presented in Table 16 andFIG. 12( b). The values are expressed as mean percent of maximal (100%)migrating cells±standard deviation. The LS PS and HS PS samples reducedPMN migration at all concentrations tested.

TABLE 16 PMN migration. Treatment Percent of Maximal Migration 1.5% LSPS 7.3 ± 1.0 0.5% LS PS 7.7 ± 0.7 0.1% LS PS 11.1 ± 0.6  1.5% HS PS 6.0± 0.8 0.5% HS PS 9.0 ± 0.4 0.1% HS PS 10.6 ± 1.3  2 ug/ml Amphotericin B9.4 ± 0.6 Untreated 13.5 ± 0.9 

Example 27 Antiinflammatory Properties of Materials from Microalgae

The pro-inflammatory cytokine interferon-gamma, which is released byactivated T-lymphocytes, plays a role in human inflammatory responses.Interferon-gamma also stimulates the enzyme indoleamine-2,3-dioxygenase,which degrades tryptophan to kynurenine. In concert with otherpro-inflammatory cytokines, interferon-gamma is the most importanttrigger for the formation and release of reactive oxygen species (ROS).Interleukin-1 alpha (IL1-α) is a cytokine that also plays a major rolein inflammatory responses. Microalgal materials were tested for theirability to suppress secretion of gamma interferon and IL1-α by humanperipheral blood mononuclear cells.

Heparinized whole blood (20-30 ml) was collected from a healthy humandonor, layered over a density gradient (Histopaque 1077 and Histopaque1119) and centrifuged at 400 g for 30 min. The PBMC rich fraction wasremoved and washed twice in with 5.0 ml RPMI-1640 without phenol redsupplemented with 5% heat-treated fetal calf serum (RPMI-FCS). Afterwashing, the cells were resuspended in RPMI-FCS at a final density of1×10⁶ cells/ml.

Fifty microliters of PBMC cells were added to wells in a 96-well plate(each treatment was tested in triplicate). Next, 50 μl of RPMI-FCSsupplemented with 2 μg/ml phytohemagglutinin (PHA) and the respectivetest materials was added to the wells. Cyclosporin A (2.5 μg/ml) wasused as a positive control, while untreated cells served as a negativecontrol (Untreated). One set of wells was not treated with PHA(Untreated-PHA). After the wells had been prepared, the plates wereincubated for approximately 68 hours at 37±2° C. and 5% CO₂. After theincubation, 20 μl of a 20:1 solution of MTS:PMS (Promega) was added toeach well and the 96-well plate was returned to the incubator for anadditional 4 hours. The plate was then read at 490 nm using a platereader. At the end of the incubation period the cell culture supernatantwas assayed for IL-1α and gamma interferon.

Prior to the assay, all buffers and reagents were prepared according tothe ELISA kit instructions (IL-1α: Cayman Chemicals (catalog number583301); γ-IFN: R&D Systems (catalog number DIF50)) and allowed to reachroom temperature. The 96-well plate was prepared by removing anyunneeded well strips and by rinsing the plate once with Wash Bufferfollowed by blotting it dry. Next, a series of IL-1α standards wasprepared ranging from 0 to 250 pg/ml and 100 μl of each of thesestandards was dispensed into two wells (duplicates) in the 96-wellplate. Subsequently, 50 μl of each sample+50 μL of culture media wasadded to additional wells (the samples were diluted to bring their IL-1αlevels within the range of the standard curve), followed by the additionof 100 μl of acetylcholinesterase:Interleukin-1α FAB′ Conjugatesolution. After reserving two empty wells to serve as blanks, the96-well plate was incubated overnight at 2-8° C. On the following daythe wells were aspirated and washed 5-6 times with Wash Buffer. Afterthe last wash was removed 200 μl of fresh Ellman's Reagent was added toeach well. The plate was incubated at room temperature with periodicchecks of the absorbance readings (405 nm) using a plate reader.

A series of gamma interferon standards was prepared ranging from 15.6pg/ml to 1000 pg/ml using human gamma interferon. Next, an ELISAmicroplate was prepared by removing any unneeded strips from the plateframe. 1001 of Assay Diluent RD1F was added to each well, followed bythe addition of 100 μl of sample (diluted in culture media if necessaryto bring the gamma interferon values within the range of the standardcurve) or 100 μl of standard per well. The microplate was covered andallowed to incubate for 2±0.25 hours at room temperature. After theincubation the plate was aspirated and washed three times with 400 μl ofwash buffer. After the last wash was removed 200 μl of anti-gammainterferon antibody/horseradish peroxidase conjugate solution was addedto each well and the plate was incubated for 1±0.25 hour at roomtemperature. The wells were aspirated and washed again as describedabove. After the last wash was removed 200 μl of horseradish peroxidasesubstrate solution (hydrogen peroxide+tetramethylbenzidine as achromagen) was added to each well and the plate was incubated for 20±5minutes at room temperature. After the incubation 50 μl of stop solution(2 N sulfuric acid) was added to each well and the plate was read usinga microplate reader at 450 nm.

As demonstrated by Tables 17 and 18 and FIGS. 13( a) and 13(b), allmaterials tested at all concentrations reduced gamma interferon andIL1-α by human PBMC cells.

TABLE 17 IL-1α assay. Treatment pg/ml 1.5% LS PS  689 ± 69.3 0.5% LS PS1530 ± 79.7 0.1% LS PS 1715 ± 69.3 1.5% HS PS  563 ± 38.5 0.5% HS PS1462 ± 99.9 0.1% HS PS 1663 ± 54.1 2.5 ug/ml Cyclosporin A 1561 ± 77.5Untreated  2402 ± 146.2 Untreated-PHA 1512 ± 88.8

TABLE 18 Gamma interferon assay. Treatment pg/ml 1.5% LS PS 1116 ± 109.70.5% LS PS 3924 ± 425.8 0.1% LS PS 4626 ± 305.5 1.5% HS PS  997 ± 159.70.5% HS PS 3509 ± 203.0 0.1% HS PS 4125 ± 131.4 2.5 ug/ml Cyclosporin A3259 ± 326.8 Untreated 11078 ± 315.0  Untreated-PHA 83 ± 7.0

Example 28 Antiinflammatory Properties of Materials from Microalgae

When presented with certain antigens, lymphocytes, which are the maincell type in PBMCs, respond by proliferating. This massive proliferationreaction forms the initial step in the immune response of this celltype. In vitro were used methods to study human lymphocyte proliferationusing the antigen phytohemagglutinin (PHA) to stimulate a proliferationresponse. The ability of microalgal materials to inhibit thisproliferation was tested.

Heparinized whole blood (20-30 ml) was collected from a healthy humandonor, layered over a density gradient (Histopaque 1077 and Histopaque1119) and centrifuged at 400 g for 30 min. The PBMC rich fraction wasremoved and washed twice in with 5.0 ml RPMI-1640 without phenol redsupplemented with 5% heat-treated fetal calf serum (RPMI-FCS). Afterwashing, the cells were resuspended in RPMI-FCS at a final density of1×06 cells/ml.

Fifty microliters of PBMC cells were added to wells in a 96-well plate(each treatment was tested in triplicate). Next, 50 μl of RPMI-FCSsupplemented with 2 μg/ml PHA and the respective test materials wasadded to the wells. Cyclosporin A (2.5 g/ml) was used as a positivecontrol, while untreated cells served as a negative control (Untreated).One set of wells was not treated with PHA and served as an index ofnon-stimulated proliferation (Untreated-PHA); all samples but thissample were treated with PHA. After the wells had been prepared, theplates were incubated for approximately 68 hours at 37±2° C. and 5% CO₂.After the incubation, 20 μl of a 20:1 solution of MTS:PMS (Promega) wasadded to each well and the 96-well plate was returned to the incubatorfor an additional 4 hours. The plate was then read at 490 nm using aplate reader.

The mean absorbance of the three wells of PBMC not treated with PHA wasdetermined and used to represent non-stimulated proliferation. Theabsorbance values for the PBMC from each treatment group were also thendetermined. A stimulated proliferation index for each treatment wascalculated using the following equations: ((Mean absorbance of treatmentwith PHA)/(Mean Absorbance without PHA))×100

The results for the PBMC proliferation assay are presented in Table 19and FIG. 18. The values are expressed as a stimulation index, using theuntreated PBMC not exposed to PHA to represent 100%. As can be seen inTable 19 and FIG. 18, all materials tested reduced PBMC proliferationcompared to PHA stimulated cells that were untreated by the testmaterials.

TABLE 19 Proliferation assay. Treatment Stimulation Index 1.5% LS PS  32± 16.0 0.5% LS PS 106 ± 6.9  0.1% LS PS 111 ± 12.0 1.5% HS PS  50 ± 13.60.5% HS PS 117 ± 14.4 0.1% HS PS 126 ± 34.4 2.5 ug/ml Cyclosporin A 123± 6.5  Untreated 183 ± 13.9 Untreated-PHA 100 ± 5.2 

Example 29 Elastase Inhibition

Human dermal fibroblasts were cultured and used as a source of theelastase enzyme. This enzyme was be partially purified from thefibroblasts by lysing the cells in an elastase buffer and retaining thesoluble portion of the lysate. Portions of this fibroblast lysate werethen be incubated with test materials a synthetic elastase substrate,Suc-(Ala₃)-p-Nitroaniline (STANA). Elastase acts upon this substrate torelease p-nitroaniline, which is detected with a spectrophotometer bymeasuring the absorbance at a wavelength of 405 nm. An inhibition of theelastase enzyme is identified by a decrease in the amount of releasedp-nitroaniline when compared to uninhibited enzyme.

Test material BM was supplied as a powder. 100 mg of BM was combinedwith either 5 ml of ethanol or 5 ml of ultrapure water in 15 mlcentrifuge tubes. After combining, the mixtures were vortexed, thenplaced onto a rocking platform for approximately 30 minutes at roomtemperature, and then centrifuged at 1,000 RPM for 5 minutes. Thesupernatants were then used at the final concentrations listed in Table20. The two additional materials used in this study, PS53 and PS133,were supplied as thick, viscous solutions (3%). These materials werealso used at the final concentrations listed in the results section.

Human neonatal dermal fibroblasts were obtained and cultured per thevendor's specifications using sterile technique at all times (CascadeBiologics, catalog #C-004-5C). The cells were seeded in a 75-cm² flaskand grown in Fibroblast Growth Medium (FGM: DMEM supplemented with 2%FBS, 5 ng/ml human recombinant growth factor, 5 ug/ml insulin, 50 ug/mlgentamicin, and 0.5 ug/ml Amphotericin-B) and subcultured until asufficient number of cells had been grown.

After washing the cells twice with PBS, approximately 7.5 ml of PBS wasadded to each culture flask and the cells were detached using a cellscraper. The detached cells were transferred to a 15 ml centrifuge tubeand the flask was rinsed with a second application of 7.5 ml of PBS,which was also transferred to the 15 ml tube. After centrifuging thetube for 5 minutes at 1,200 RPM (4° C.), the supernatant was removed anddiscarded while the pellet was resuspended in 2 ml of ice cold 2×elastase buffer (400 mM Tris-HCl [pH 8.0], 0.2% Triton X-100) andsonicated 3× for 10 seconds or until the lysate became clear. The lysedcells were centrifuged at 2,200 RPM for 10 minutes (4° C.) to remove anycellular debris. The supernatants from all of the preparations werepooled into a single container, mixed, and then aliquoted into 2 mlportions and stored at −75±5° C. until used. One of the aliquots wasused to determine the protein concentration (BCA Protein Assay) andelastase activity level of the batch.

A Bicinchoninic Acid assay (Pierce, Inc., BCA Protein Assay Kit, catalog#23227) Fifty volumes of Reagent A (Bicinchoninic Acid BCA was combinedwith 1 volume of Reagent B (4% (w/v) CuSO₄-5H₂O) in a 15-ml centrifugetube. For the assay, proteins can reduce Cu(II) to Cu(I) in aconcentration dependent manner. BCA can then react with the Cu(I) toform a purple colored complex with a maximum absorbance at 562 nm. Twohundred microliters of this combined reagent was dispensed into a96-well plate. Next, 10 μl of each of the standards was added into theirrespective wells (standards were made using 2 mg/ml bovine serum albumindissolved in PBS, and then making a series of 50% dilutions down to0.0078 mg/ml). Next, 10 μl of 2× elastase buffer (used as a blank) orcell lysate sample was added. The plate was covered and incubated at37±2° C. for 30±5 minutes. At the end of the incubation the absorbanceof each well was measured at 540 nm using a microplate reader.

After measuring the protein concentration of the lysate, a small samplewas obtained and the elastase activity of the lysate was determined at100%, 50% and 25% concentrations using 2× elastase buffer to make thedilutions. To determine the activity, 100 μl of the three differentlysate concentrations was loaded into a 96 well plate (eachconcentration tested in triplicate), while 100 ul of 2× Elastase bufferwas added to three additional wells to serve as blanks. Next, 100 μl ofdeionized water was added to all wells and the 96-well plate will beallowed to incubate with the samples for 15 minutes at 37±2° C. Next, 4μl of STANA (62.5 mM Suc-(Ala₃)-p-Nitroaniline in DMSO) was added toeach well, and the 96-well plate was returned to the incubator for 1hour. After the incubation the well plate was read at 405 nm using amicroplate reader. The mean absorbance for each concentration of thelysate was plotted versus its respective concentration. These valueswere used to estimate a concentration that will produce aspectrophotometric measurement of 0.4 to 0.5 using a linear regressionanalysis of the best-fit line through all three (100%, 50%, and 25%)data points. The stored fibroblast lysates was diluted to theappropriate concentration to elicit the desired level of activity.

Test materials were prepared at 2× their final desired concentration indeionized water. If solvents were present in the test materials thenappropriate solvent controls were also prepared. 100 μl of distilledwater served as a negative control while 100 μl of 0.2 mM Phosphoramidonserved as a positive control. After all of the wells had been preparedthe well plate was sealed with an adhesive cover and incubated for 15minutes at 37±2° C. to allow the inhibitors time to interact withelastase. After this preliminary 15 minute incubation, 4 μl of STANA wasadded; the plate was resealed and then incubated for 1 hour at 37±2° C.At the end of the incubation the plate was read at 405 nm using amicroplate reader.

TABLE 20 Anti-elastase assay. Treatment Percent Inhibition  10% BM waterextraction 59   5% BM water extraction 40  10% BM Ethanol extraction 28  5% BM Ethanol extraction 16   1% LS PS 29 0.5% LS PS 23   1% LS PS 310.5% HS PS 19 100 μM Phosphoramidon 94

Example 30

Microalgal materials were tested to assess their ability to relieveredness and pain associated with sunburn. Informed consent was obtainedfrom human subjects. A 1.5% preparation of polysaccharide fromPorphyridium cruentum, purified essentially as described in Example 2,was formulated with 1% Germaben II and 0.15% sodium EDTA. A patch of armskin was exposed to UV radiation from a Solar Ultraviolet Simulatormodel 14S with a dose controller system model DCS-1 and a 150 watt xenonarc lamp #1122200 (Hanovia) having a continuous emission spectrum in theUV-B range of 290-320 nanometers. The dose was tested on each subjectprior to the experiment to determine the minimal erythema dose (MED)necessary to achieve a level 2 response in each subject. Erythema wasscored by a trained evaluator using the following scale: 0=no reaction;+=minimal erythema; 1=slight erythema; 2 moderate erythema; 3=markederythema with or without edema; 4=severe erythema with or without edema.Subjects wer them exposed to a UVB dose sufficient to generate a level 2erythema at three sites on the skin. One site was treated with thepolysaccharide before and after irradiation. One site was treated withpolysaccharide only after irradiation. One site was left untreated as acontrol. After 4, 24, 48, 72, and 96 hours, follow-up examination wasperformed. Polysaccharide was applied once daily to the two treatedsites following daily examinations and was also treated once per day athome by each subject.

Mean erythema scores were the same on all three sites tested through the4 and 24 hour evaluation points. At the 48 hour evaluation the meanerythema score decreased 5% on the site treated pre and post irradiationand stayed the same on the other two sites. At the 72 hour evaluationthe mean erythema scores decreased by 30% on both sites treated with thepolysaccharide and decreased 13% on the untreated site. At the 96 hourevaluation the mean erythema scores decreased by 40% on both sitestreated with the polysaccharide and decreased 22% on the untreated site.There was no edema observed at any time on any site. The polysaccharidetreatment was associated with a clinically significant decrease inerythema compared to no treatment.

Example 31

Polysaccharide was prepared essentially as described in Example 2 wastested for the ability to protect a skin model from TT dimer formation.The MatTek EpiDerm® tissue skin model was generate as described inExample 23, as well as polysaccharide application and UV exposure.

After fixing the tissues overnight in 10% neutral buffered Formalin thetissues were processed as follows: 1. tissues were washed in 80%ethanol, (15-20 minutes); 2. tissues were washed in 95% ethanol, (3times, 15-20 minutes); 3. tissues were washed in Absolute ethanol (3times, 15-20 minutes); 4. tissues were washed in 50% absoluteethanol/50% xylene (15-20 minutes); 5. tissues were washed in Xylene (2times, 15-20 minutes each); 6. tissues were washed in Paraffin (3changes, 15 minutes each); 7. tissues were embedded and sectioned (4-6mm sections) onto microscope slides.

Immunohistochemistry: Tissue Hydration and Pre-ImmunohistochemicalProcessing: 1. tissues were washed with Xylene (3 times, 5 minuteseach); 2. tissues were washed with Absolute ethanol (2 times, 10 minuteseach); 3. tissues were washed with 95% ethanol (2 times, 10 minuteseach); 4. tissues were washed with 80% ethanol (1 time, 5 minutes); 5.tissues were washed with Distilled water (1 time, 1 minute); 6. Antigenunmasking was performed by washing with 10 mM sodium citrate buffer (pH6.0) at 95° C. for 10 minutes and then rinsing in distilled water 3times, 2 minutes each; 7. endogenous peroxide activity was quenched(0.5% hydrogen peroxide for 5-10 minutes and then rinse in PBS 2×, 5minutes each).

Immunohistochemistry: Immunoperoxidase Staining: 1. sections wereincubated for 1 hour in 1.5% goat serum in PBS. 2. primary antibody(anti-thymine dimer antibody, clone KTM53, Kamiya Biomedical Company)was incubated for 30 minutes at room temperature or overnight at 4° C.Primary antibody was diluted in 1.5% goat serum in PBS. 3. Sections werewashed 3× with PBS, 5 minutes per wash. 4. Sections were incubated for30 minutes with biotin-conjugated secondary antibody (goat anti-mouse,Santa Cruz Biotechnology). Secondary antibody was diluted in 1.5% normalblocking serum. 5. sections were washed 3× with PBS, 5 minutes per wash.6. Sections were incubated for 30 minutes with the avidin/HRP enzymecomplex (ABC Staining System, Santa Cruz Biotechnology) and then washthe sections 3× with PBS, 5 minutes per wash. 7. Sections were incubatedin HRP substrate solution for about 5 minutes. 8. Sections were washed1× in distilled water for 5 minutes. 9. sections were counter-stained inhematoxylin for 5-10 seconds and then rinsed through several changes ofdistilled water. 10. Sections were dehydrated as follows: a. sectionswere washed in 95% ethanol (2×, 10 seconds each); b. sections werewashed in 100% ethanol (2×, 10 seconds each) c. sections were washed inxylene (3×, 10 seconds each). Mounting medium (Shurmount, TriangleBiomedical Sciences, Inc.) was applied, followed by a microscope coverslip.

Example 32

Polysaccharide was tested on human volunteers obtained for various skinparameters. The study was conducted in compliance with CFR Title 21,Part 50 (Informed Consent of Human Subjects). Informed Consent wasobtained from each subject in the study and documented in writing beforeparticipation in the study. Twenty-seven (27) female subjects ranging inage from 38 to 60 years were impaneled for the 12 week testingprocedure.

Subjects reported to the testing facility for a baseline visit. Atrained technician evaluated the presence of crow's feet finelines/wrinkles on the face of each subject and evaluated pore size andskin texture. The elasticity of the skin was measured using theCutometer® (Courage+Khazaka, Germany) and the moisture content of theskin was measured using the Corneometer® (Courage+Khazaka, Germany).Skin replicas were taken at the crow's feet area to evaluate finelines/wrinkles and on the cheek to evaluate pore size. Subjects weregiven a 1.5% (w/w) polysaccharide solution prepared essentially asdescribed in Example 2, which also contained 1% (w/w) Germaben II® and0.15% (w/w) disodium EDTA.

Subjects applied a sufficient quantity of polysaccharide to cover theentire face, including eye area, in the morning and evening, and rubbedthe polysaccharide in until it was completely absorbed by the skin. Thetwice daily application occurred for a period of 12 weeks.

Fine lines and wrinkles/crows feet were evaluated reduced by 3.1% after4 weeks, 4.6% after 8 weeks, and 3.1% after

Skin elasticity, measured by Cutometer®, demonstrated an average 4.4%increase in elasticity after 8 weeks and an average 3.4% increase inelasticity after 12 weeks.

Skin moisturization level, measured by a Comeometer®, increased anaverage of 7.4% after 8 weeks, and 8.8% after 12 weeks. Skinmoisturization level was also measured 15 minutes after the initialapplication, and averaged an increase of 4.2%.

Transepidermal water loss was measured using a Tewameter®. Averagedecrease in transepidermal water loss was 13.9% after 12 weeks.

Additionally, skin replica analysis indicated a smoothing effect incoarse lines as well as a reduction in the number. 76% of subjectsresponded their skin was less rough and 68% felt that the overallquality of their skin improved. 81% of subjects responded that theirskin felt tighter immediately after application, and 85% of subjectsresponded that their skin felt tighter 30 minutes after application.

Example 33

Glucose transporters and a gene encoding a fusion of the Porphyridiumpolysaccharide-binding protein:ble protein were cloned into pBluescriptand were used to transform Porphyridium sp.

Plasmid Constructs: All the plasmid constructs were designed to have theexpression of the heterologous gene driven by endogenous promoter Glp(which drives the expression of the glycoprotein gene). The GlpBleconstruct has the Ble gene cloned as a fusion downstream of the Glpgene.

The architecture of the constructs is as follows:

Plasmid 1: Ble: (SEQ ID NO: 11) 5′ Glp:: Ble::cmv 3′ Plasmid 2: HumanGlut1 (HuGlut): 5′ Glp:: HuGlut::cmv 3′ Plasmid 3: Chlorella hexose(ChlHex): 5′ Glp:: ChlHex::cmv 3′ Plasmid 4: Yeast Hxt2 (YeHxt2): 5′Glp:: YeHxt2::cmv 3′ Plasmid 5: Ble Fused to GLPcDNA (GLPBle): 5′ Glp::GLPBle::cmv 3′

GLP:BLE is (SEQ ID NO:15:SEQ ID NO:11); 5′ Glp is the glycoproteinpromoter (SEQ ID NO:21); Transporter sequences are documented inapplications cited herein, such as PCT Patent application No:PCT/US2007/001319, filed Jan. 19, 2007, entitled “NutraceuticalCompositions from Microalgae and Related Methods of Production andAdministration”. Cmv 3′ is the 3′UTR listed in SEQ ID NO:32.

Biolistic Transformation: All transformations were performed asco-transformations using plasmid 1 in combination with one of plasmids2-5. The biolistic particle delivery system PDS1000/He (Bio-Rad, USA)was used for transformation. Porphyridium sp. culture was grown tologarithmic phase (˜2×10⁶ cells/mL) in ASW (ATCC) media, undercontinuous light (˜75 umol/m²). Cells from this culture were harvestedat 4000 rpm at room temperature. The cell pellet was washed twice withsterile distilled water. Cells were resuspended in fresh ASW (ATCC)media to the same cell density i.e. ˜2×10⁶ cells/mL and incubated in thedark for 16 hours. The dark adapted cells were then harvested at 4000rpm at room temperature, resuspended to a density of ˜2×10⁸ cells/mL infresh ASW (ATCC) media. About ˜1×10⁸ cells were transferred to eachplate. The plasmid DNA was coated onto 550 nm gold particles (S04e,Seashell Technology, Inc). Ten uL gold particles coated withco-transforming plasmid DNA were delivered to each plate using a 1350psi rupture disc, under a vacuum of 28 in. Hg. The transformed cellswere recovered for a day in non selective medium, and then harvested,resuspended in ASW (ATCC) media and transferred to ASW (ATCC) agarplates supplemented with 15 ug/mL Zeocin. These plates were incubatedunder 75 umol/m² light conditions for 10-14 days. Colonies growing onthese plates were transferred to fresh ASW (ATCC) plates supplementedwith 15 ug/mL Zeocin. The resistant colonies were selected for furtheranalysis.

Genotyping: Genotyping was done on genomic DNA extracted from WT, thetransformants and plasmid DNA. For each co-transformation set, seventransformants (numbered 1 to 7) were genotyped for the resistant marker,ble and the corresponding co-transforming plasmid. The following primersets were used to genotype for the different plasmids:

Construct Primer set (SEQ ID NOs) Ble SEQ ID NO: 49 + SEQ ID NO: 48 Ble2SEQ ID NO: 47 + SEQ ID NO: 48 HuGlut1 SEQ ID NO: 49 + SEQ ID NO: 50 ChlHex SEQ ID NO: 49 + SEQ ID NO: 51 Ye Hxt2 SEQ ID NO: 49 + SEQ ID NO: 52GLPBLE SEQ ID NO: 53 + SEQ ID NO: 48

The PCR profile used was as follows: 94° C. denaturation for 5 min; 35cycles of 94° C.-30 sec, 58° C. or 62° C. or 66° C. (58° C. for ble2,62° C. for GLPBle and 66° C. for ble, HuGlut and Chl Hex,)-30 sec, 72°C.-2 min; 72° C.-5 min.

Results are shown in FIG. 20. In each of the four panels, the top gelshows genotyping of individual transformants for the ble gene (plasmid1), while the bottom gel shows genotypoing of the same transformants forplasmids 2-5. P is plasmid DNA template showing the expected size of thegenotyping PCR product; W is wild type DNA. 1-7 are 7 independenttransformants. Panel (a) shows integration of plasmid 2 into the genomeof 6 transformants. Panel (b) shows integration of plasmid 4 into thegenome of 3 transformants. Panel (c) shows integration of plasmid 3 intothe genome of 5 transformants. Panel (d) shows integration of plasmid 5into the genome of 4 transformants.

TABLE 21 Genbank accession numbers of superoxide dismutases. CAA42737;AA43859; AA29934; AA02655; P_625295; H004200; AC14367; P_001003721;BM60577; AM08755; P_966270; P_963716; ABM37237; ABM35060; ABM33234;AAY48900; NP_000445; AAH12423; AAA62278; ABM33141; NP_407488; NP_405922;YP_932026; ZP_01641300; ZP_01638301; ZP_01637188; EAX24391; EAX23794;EAX23720; EAX23627; EAX20859; EAX19390; EAX16596; CAL93139; YP_914326;YP_747424; ABI59459; ZP_01610569; ZP_01605216; ZP_01600343; ZP_01584712;ZP_01581863; ZP_01581157; ZP_01575777; ZP_01569848; ZP_01559998;EAW01367; EAW01065; EAV97274; EAV95856; EAV80568; EAV73624; EAV73531;EAV70130; EAV66456; EAV61854; ZP_01532079; ZP_01516088; EAV26209;YP_845889; YP_822355; YP_843115; YP_836186; and ABK17454

TABLE 22 Genbank accession numbers of beta-carotene hydroxylases.NP_682690; YP_172377; NP_440788; YP_475340; YP_475340; BAB75708;ZP_01084736; ZP_01080915; ZP_01123496; ABB27016; NP_895643; NP_896386;ABB34062; YP_292794; AAP99312; ZP_01006041; ABB49299; NP_848964;ZP_01040435; NP_049051; YP_457405; and AAT38625

All references cited herein, including patents, patent applications, andpublications, are hereby incorporated by reference in their entireties,whether previously specifically incorporated or not. The publicationsmentioned herein are cited for the purpose of describing and disclosingreagents, methodologies and concepts that may be used in connection withthe present invention. Nothing herein is to be construed as an admissionthat these references are prior art in relation to the inventionsdescribed herein.

Although this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

1. A gel, lotion, cream or ointment composition suitable for topicaladministration comprising microalgal exopolysaccharide particles, theexopolysaccharide produced by microalgae of the genus Parachlorella,wherein the exopolysaccharide particles are made by: culturingmicroalgae cells in a culture medium so that exopolysaccharide issecreted by the microalgal cells into the culture medium; separating theexopolysaccharide from the microalgal cells to create a purifiedexopolysaccharide; heating the purified exopolysaccharide at atemperature of 148-160° C. so as to render it partially insoluble inwater, and so that it forms a polysaccharide gel in water; andgenerating particles having an average size of between 0.1 and 400microns from the heated exopolysaccharide, wherein the particlesincrease in volume on contact with water by at least 100% compared totheir anhydrous volume; and wherein the particles increase in volume forat least 4 hours.
 2. The composition of claim 1, wherein the particlesincrease in volume on contact with water by at least 200% compared totheir anhydrous volume.
 3. The composition of claim 1, wherein thecomposition further comprises between 50-97% ethylhexyl palmitate, 1-5%silica dimethyl silylate, 0.1-3% butylene glycol, and 0.1-3% of one ormore preservative selected from the group consisting of diocide, dowicil200, and methyl paraben.
 4. The composition of claim 1, wherein theexopolysaccharide contains at least 3.0% sulfur by weight.
 5. Thecomposition of claim 1, wherein the particles are generated by millingthe heated purified exopolysaccharide.
 6. The composition of claim 1,further comprising at least one ingredient selected from the groupconsisting of beta carotene, lutein, astaxanthin, vitamin C, vitamin E,vitamin A, and coenzyme Q10, a peptide, an acetylated peptide, oilsoluble α-hydroxy acid, an alkyl lactate, and salicylic acid.
 7. Thecomposition of claim 6, wherein the microalgal exopolysaccharide and theat least one ingredient have been subjected to heating, drying andhomogenization to form particles comprising both microalgalexopolysaccharides and the at least one ingredient.
 8. The compositionof claim 7, wherein the particles comprising both microalgalexopolysaccharides and the at least one ingredient have been milled.